Review ArticleDengue Fever Causes Complications and Vaccine Strategies
Niyati Khetarpal12 and Ira Khanna1
1 International Centre for Genetic Engineering and Biotechnology Aruna Asaf Ali Marg New Delhi 110067 India2Department of Biochemistry University of Delhi Institute of Home Economics Hauz Khas New Delhi 110016 India
Correspondence should be addressed to Niyati Khetarpal 27niyatigmailcom
Received 14 March 2016 Revised 18 May 2016 Accepted 1 June 2016
Academic Editor Mario Clerici
Copyright copy 2016 N Khetarpal and I KhannaThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Dengue is a highly endemic infectious disease of the tropical countries and is rapidly becoming a global burden It iscaused by any of the 4 serotypes of dengue virus and is transmitted within humans through female Aedes mosquitoesDengue disease varies from mild fever to severe conditions of dengue hemorrhagic fever and shock syndrome Globalizationincreased air travel and unplanned urbanization have led to increase in the rate of infection and helped dengue to expandits geographic and demographic distribution Dengue vaccine development has been a challenging task due to the existenceof four antigenically distinct dengue virus serotypes each capable of eliciting cross-reactive and disease-enhancing antibodyresponse against the remaining three serotypes Recently Sanofi Pasteurrsquos chimeric live-attenuated dengue vaccine candidatehas been approved in Mexico Brazil and Philippines for usage in adults between 9 and 45 years of age The impact of itslimited application to the public health system needs to be evaluated Simultaneously the restricted application of this vaccinecandidate warrants continued efforts in developing a dengue vaccine candidate which is additionally efficacious for infantsand naıve individuals In this context alternative strategies of developing a designed vaccine candidate which does not allowproduction of enhancing antibodies should be explored as it may expand the umbrella of efficacy to include infants and naıveindividuals
1 Introduction to Dengue
(1) Overview Dengue is an infectious disease caused byany of the four dengue virus serotypes DENVs 1ndash4 It isa mosquito-borne disease and is primarily transmitted tohumans by the female Aedesmosquito The disease is mainlyconcentrated in tropical and subtropical regions puttingnearly a third of the human population worldwide at risk ofinfection [1] Infection with DENV results in varying degreesof pathological conditions ranging from mild asymptomaticdengue fever (DF) to severe dengue hemorrhagic fever (DHF)and dengue shock syndrome (DSS) which may turn fatal [2]A dramatic worldwide expansion of the DENV has occurreddue to rapid urbanization increase in international travellack of effectivemosquito controlmeasures and globalization[3] Though there is no approved drug an update by SanofiPasteur reveals licensure of its vaccine in Mexico BrazilPhilippines and El Salvador [4]
(2) Epidemiology Dengue has become one of the most wide-spread reemerging mosquito-borne diseases globally Inci-dence of dengue has increased 30-fold in last five decades[5] Currently dengue is endemic to 128 countries mostlydeveloping nations posing a risk to approximately 397billion people annually A recent dengue distribution modelhas estimated 390 million dengue infections annually out ofwhich 96million cases occurred apparently [6 7]The Indiansubcontinent is the epicenter of dengue [8] with cases beingheavily underestimated [9] Thus there is an urgent need ofimprovement in serosurveillance to enable the authorities toprepare adequately for an outbreak
(3) Vector Dengue viruses are transmitted in humans byfemale Aedes (Ae) mosquitoes of the subgenus StegomyiaAe aegypti has been the most important epidemic vector inthe tropical and subtropical regions Other species such asAe albopictus Ae polynesiensis member of Ae scutellaris
Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2016 Article ID 6803098 14 pageshttpdxdoiorg10115520166803098
2 Journal of Immunology Research
ER lumen
Cytoplasm
Pr (10)
C (12)
E (60)
M (9)
NS1 (48)
NS2A (20)
NS2B(145)
NS3 (70)
NS4A(16)
NS4B (27)
NS5 (105)
Signalase
Furin
Unknown
NS2B-3 protease
ER membrane
C998400
N998400
Figure 1 Genome organization and membrane topology of dengue virus The viral RNA is translated as a single polyprotein consistingof structural (light brown-C prM and E) and nonstructural (dark brown-NS1 2A 2B 3 4A 4B and 5) protein components Symbols CprM E NS and PM denote capsid protein precursor membrane protein envelope protein nonstructural proteins and plasma membranerespectivelyThis single polyprotein then gets processed by viral (green arrow) and host (black arrow) proteasesThe structural proteins (prMand E) remain anchored on the luminal side of the ER membrane The C protein is anchored on the cytoplasmic side of ER membrane prMis later cleaved by furin (red arrow) in the TGN into the pr peptide and M protein The NS proteins are mainly processed by NS2B-NS3(viral protease) in the cytoplasm NS2A2B and NS4A4B are transmembrane proteins and thus stay anchored in the ER The approximatemolecular weight (in kDa) of each protein has been indicated in braces
complex and Ae niveus have been found to play a roleas secondary vectors [8] However Ae niveus is consideredonly as a sylvatic vector The life cycle of Aedes mosquitodepending upon the extent of feeding lasts for 8ndash10 days atroom temperature It consists of two phases aquatic (larvaepupae) and terrestrial (eggs adults) phase Presently Aealbopictus has become an increasingly important vector as itcan easily adapt to new environments including temperateregions Its spread to Ae Aegypti free countries has createdopportunities for dengue viruses to enter new locations andcause disease [10] However it is still a minor contributor tohuman dengue infections
2 Dengue Virus
21 Genomic Structure The viral genome consists of a posi-tive sense RNA of sim11 kb This RNA is translated into a singlepolyprotein which encodes for three structural proteinsnamely capsid (C) premembrane (prM) envelope (E) and 7nonstructural proteins (NS1NS2ANS2BNS3NS4ANS4Band NS5) (Figure 1)
It consists of a single open reading frame and two non-coding regions (NCRs) at the 51015840 and 31015840 ends It is expressedas a single polyprotein precursor which is coposttransla-tionally cleaved by viral and host proteases (Figure 1) The51015840 and 31015840 NCRs contain secondary structures and conservedsequences which are involved in regulation of viral repli-cation The 51015840UTR (sim100 nucleotides) has a type I methy-lated cap structure (m7G51015840ppp51015840A) but the 31015840UTR (sim450nucleotides) lacks a terminal polyadenylate tail Proteinsynthesis occurs in the cytoplasm on the Rough Endoplasmic
Reticulum (RER) and the structural proteins get anchored tothe ER on the luminal side where assembly and maturationof virion occur (Figure 1) [2 11] Major functions of all theproteins are summarized in Table 1 [11 12]
22 Structure of Virion and Envelope Protein A three-dimen-sional image reconstruction of mature dengue virus showsthat it is sim50 nm in diameter and consists of an outer proteinshell (E and M) a lipid bilayer and a less characterizednucleocapsid core (C and RNA genome) Dengue virusexhibits different surface structures during itsmaturation andinfection and these conformational changes are attributed tothe inherent flexibility of the envelope protein E protein ismade up of three domains namely EDI (red) EDII (yellow)and EDIII (blue) and transitions between its oligomericstates are supported by the hinge motion that occurs betweenEDI-EDII and EDI-EDIII The immature virus particle hasa spiky appearance with 60 trimeric surface spikes eachconsisting of three prM-E heterodimers (Figure 2) The prpeptides are cleaved in the TGN by furin which leads torearrangement of the E proteins into 90 homodimers of E thatlie flat against the viral surface giving a smooth appearance acharacteristic ofmature virus In an E dimer the Emonomersare arranged face to face with their long directions beingantiparallel to each other M remains anchored to the lipidbilayer below the E protein shell (Figure 2)
When mature virus infects a new cell through receptor-mediated endocytosis the E protein molecules shift to atrimeric conformation in the acidic compartment of an endo-some protrude from the virus surface causing membranefusion and facilitate viral RNA release into the cytoplasm(Figure 2) [2 13 14]
Journal of Immunology Research 3
Table 1 Functions of DENV proteins
Protein FunctionStructural
Capsid (C) Binds and stabilizes viral RNA
Premembranemembrane (prMM)(i) Pr peptide functions as cap that protects the fusion peptide on E thus preventingpremature fusion(ii) M forms ion channel
Envelope (E) (i) Recognition and binding to the host cell(ii) Involved in uncoating of virus by enabling fusion of viral and endosomal membranes
Nonstructural (NS)NS1 (i) Viral RNA replication
(ii) Viral defense through inhibition of complement activationNS2A Viral replication and assemblyNS2B NS3 protease cofactorNS3 (i) Serine protease-cleaves viral polyprotein
(ii) RNA helicase and RTPaseNTPase-viral RNA replication(iii) Induction of apoptosis in infected cells
NS4A Induces membrane alterations and autophagy to enhance virus replicationNS4B (i) Interacts with NS3-viral replication
(ii) Blocks IFN-120572120573-induced signal transduction and helps virus to escape hostrsquos innateimmune response
Figure 2 Organization of E protein on dengue virus surface duringits life cycle The E protein is colored as follows EDI (red) EDII(yellow) EDIII (blue) and the FL (green) prM and M proteinare colored as cyan (a) Immature virus contains 60 trimericspikes of E and prM heterodimer (b) Mature virus contains 90homodimers of E protein (c) These homodimers then furtherundergo reorganization to form fusion active E homotrimers inwhich fusion loop is exposed M protein is not shown in the fusiontrimer for simplicity E C M FL and prM denote envelope capsidmembrane fusion loop and precursor membrane respectively
The E protein is the major exposed antigen of the denguevirion antibodies against which provide immunity duringnatural infectionThe E proteins of the four DENV serotypeshave 60ndash70 amino acid similarity and are glycosylated atAsn-67 (unique to dengue) and Asn-153 These residues havebeen found to play important roles in the receptor attachmentand viral entry into the cell The E protein consists of atransmembrane region and an ectodomain which is dividedinto three structuralfunctional domains [15]
(i) EDI (envelope domain I) central region contains 8-stranded 120573-barrel and organizes the structure
(ii) EDII (envelope domain II) is a dimerization domainand contains 12 120573 strands 2 120572 helices and a highlyconserved fusion loop
(iii) EDIII (envelope domain III) contains immunoglob-ulin like domain with 10 120573 strands and is involved inreceptor binding
Apart from the primary difference in their structure the threedomains of the ectodomain differ in their immunogenicity asillustrated schematically in Figure 3 The EDIII of each of thefour serotypes (circled and colored in red green blue andblack for DENV-1 DENV-2 DENV-3 and DENV-4 resp)elicits strongly neutralizing antibodies which are largelyserotype-specific [16] Strongly neutralizing serotype-specificantibodies have been largely found to be elicited againstthe EDIII hinge region [17] complex quaternary epitopesdisplayed on the E protein dimer and thewhole virion [18 19]
4 Journal of Immunology Research
Figure 3 DENV-1 DENV-2 DENV-3 and DENV-4 with EDIII(circled) in red green blue and black respectively EDIIIs elicitstrongly neutralizing serotype-specific antibodies (antibodies inbold) Owing to homology especially in domains (EDIII and prM)in yellow cross-reactive weakly neutralizing antibodies (unboldantibodies) and nonneutralizing enhancing antibodies (dashedantibodies) are elicited in bulk
It has been reported that it is serotype-specific neutralizingantibodies and not cross-reactive neutralizing antibodiesthat confer protection against infection [20] It has beenreported that bulk of the immune response is elicitedagainst the cross-reactive domains of EDI EDII and prM(yellow domain in the virus image) [16 21] Anti-EDIIIantibodies (Figure 3 unbold antibodies) are largely het-erotypic weaklynonneutralizing while prM antibodies (Fig-ure 3 dashed antibodies) are largely nonneutralizing andcross-reactive It is believed that the virus utilizes suchweaknonneutralizing cross-reactive antibodies in gainingaccess into the host cell via Fc receptor as an alternativepathway during a secondary infection with a heterologousserotype leading to enhancement of infection [16 20 21]This phenomenon is known as antibody-dependent enhance-ment (ADE)
Thus it can be inferred that strongly neutralizing anti-bodies prevent ADE caused by a larger population of weaklynonneutralizing enhancing antibodies A vaccine candidatecapable of a larger population of strongly neutralizing anti-bodies could probably be an ideal vaccine showcasing strongprotection without ADE This can probably be achieved bydesigning the vaccine candidate and not by default strategy
23 Cellular Targets and Receptor Interaction The currentmodel of flavivirus cell entry suggests the use of two func-tionally different sets of molecules attachment factors thathelp the virus to concentrate on the cell surface and primaryreceptor(s) which help directing the virion to the endocyticpathway (Figure 4) [22]
During natural dengue infection in humans themosquito delivers virus in skin epithelium where it infectsand replicates in the cells of mononuclear lineage like mono-cytes dendritic cells macrophages and Langerhans cells[23 24] These infected cells carry the virus to lymph nodeswhere it replicates resulting in viremia which is followedby systemic infection of liver lungs and spleen Howeverin mosquitoes the primary target of DENV infection is theepithelium of the midgut where it first replicates and then
[25 26] spreads to and replicates in salivary glands fromwhere the infection is transmitted through saliva to the nextvertebrate host during the blood meal
3 Dengue Disease
31 Classification The spectrum of clinical illness may rangefrom asymptomatic disease to a broad range of syndromeswith severe clinical manifestations Symptomatic infectionmay range frommild debilitating DF to life threatening DHFand DSS due to plasma leakage in DHF patients Thesethree conditions likely represent progressively severe stagesof a continuous dengue disease spectrum [27] They arebased on traditional WHO classification case definitions andcontinue to be recognized in many regions of the worlddespite the introduction of a new classification system Thenew classification based on a single parameter [5] allowsbetter case capture [28] but is not compatible with restrictedhealth care facilities in endemic regions especially duringoutbreaks [29]
32 DF DF is a self-limiting fever lasting usually for 5ndash7days It is sometimes debilitating during the acute illnessstage The clinical features of DF vary according to the ageof the patient The infants and young children may haveundifferentiated febrile sickness with maculopapular rashThe older children and adults may have mild febrile syn-drome or severe disease with high fever (usually bipha-sic) severe headache retroorbital pain myalgia arthralgianausea vomiting and petechiae Leukopenia and thrombo-cytopenia are usually observed in all ages In some casesDF may accompany bleeding complication such as gingivalbleeding epistaxis gastrointestinal bleeding haematuriaand menorrhagia (in case of women) [27]
33 DHFDSS DHF is characterized by symptoms of DFalong with thrombocytopenia hemorrhagic manifestationsand plasma leakage A positive tourniquet test may be sug-gestive of DHF however this is being debated now due to itslow sensitivityspecificity Plasma leakage determines diseaseseverity in DHF It is also the most important differencebetween DHF and DF Depending on disease severity andclinical manifestations DHF is divided into four grades Ito IV with grade IV being the most severe Several patientsalso have fine petechiae scattered on the extremities axillaeface and soft palate usually seen in the febrile periodThe critical phase is usually reached at the end of febrileillness marked by rapid decrease in temperature and oftenaccompanied by circulatory disturbances including plasmaleakage hemoconcentration and thrombocytopenia [27]
In severe cases with critical plasma loss DSS ensuesand may be life threatening if not treated properly DSS ischaracterized by a rapid weak pulse with narrowing pulsepressure (lt20mmofHg) cold clammy skin and restlessnessThe patient may die within 12ndash24 h of going into shock orrecover rapidly with volume replacement therapy
34 Primary and Secondary Dengue Infection The first expo-sure of an individual to any of the four dengue virus serotypes
Journal of Immunology Research 5
Mature dengue virion
GRP78Mannose receptor
Heparan sulfate
TAMTIM
HSP 7090NKp44DC-SIGN
Clathrin coated pit Internalisation via endocytic pathway
(A) (B)
PM
Outside
Cytoplasm
Figure 4 Schematic representation of the dengue virus entry process The dengue virus makes use of membrane receptors and attachmentfactors on the cell plasma membrane (PM) to find its way to the cytoplasm The mature virion either gets attached directly to a cellularmembrane receptor (a) or uses several attachment factors (b) to finally trigger the endocytic clathrin dependent pathway The endocyticvesicle becomes a late endosome where acidification triggers conformational changes on the E protein dimers to become fusogenic trimersFinally pores are formed and the genome of the virus is released into the cytoplasm
is known as primary dengue infection It maymay not resultin symptomatic infection In primary infection high titersof immunoglobulin M (IgM) and immunoglobulin G (IgG)antibodies appear in 3ndash5 and 6ndash10 days respectively afterthe onset of infection The presence of IgM is transientdisappearing in 2-3 months after the onset of illness whereasIgG persists for life [30] Hence primary infection with aparticular serotype provides life-long immunity against thatserotype But it does not provide continued cross-protectiveimmunity against the remaining serotypes
A secondary infection with a previously unencounteredDENV serotype usually results in classical DF However 2-3of secondary infection cases develop intoDHFwhichmayprogress to DSS and death During a second infection with adifferent serotype the presence of low amounts of heterotypicantibodies (which form complexes with DENVs) promotesthe access of the virus to monocytes via Fc receptors leadingto an increase in viral load and severity of the disease Thisphenomenon is known as ADE The major players of thisphenomenon are cross-reactive antibodies elicited againstthe fusion loop and prM which are found to be weaklyneutralizing leading to enhancement of infection at lowconcentrations [16 21] Although ADE has been found toresult in disease severity all the severe cases are not associatedwith secondary infection nor do all the cases of secondaryinfection progress to DHFDSS [2] In addition to humoralimmunity cross-reactive memory T cells could also playa role in either providing protective immunity or causingimmunopathology [31]
35 Diagnosis and Clinical Management Dengue infectionis usually confirmed by identification of viral genomic RNAantigens or the antibodies it elicits Antigen detection testsbased on NS1 detection have been designed to detect thedengue viral NS1 proteinwhich gets released from the dengueinfected cells and appears early in the bloodstream A 3-in-1test for simultaneous detection of NS1 IgM and IgG is nowavailable ELISA-based serological tests are easy to performand are cost-effective for dengue detection
Up to date there is no antiviral drug available fordengue Treatment is usually based on symptoms and isperformed through medical support For uncomplicatedcases of dengue fever the treatment prescribed is bed restoral rehydration and paracetamol as an antipyretic andanalgesic Patientrsquos health is monitored through variousblood tests from fever day 3 onwards till the conditionimproves Clinical signs that signal progression to seriousdisease include cold limb extremities low pulse low urineoutput signs of mucosal bleeding and abdominal pain DHFis indicated by a rising hematocrit (ge20) and a fallingplatelet count (gt100000mm3) If any of these signs aredetected immediate hospitalization is necessary Treatmentfor DHF patients is based on intravenous fluid therapy tomaintain effective circulation during plasma leakage pluscareful clinical monitoring of hematocrit platelet countpulse rate and blood pressure temperature urine outputfluid administered and other signs of shock Patients usuallyrecover within 12ndash48 h of fluid therapy Treatment for DSSpatients mainly consists of immediate fluid therapy with
Figure 5 Classification of dengue vaccine candidates
colloids and extensive monitoring of any complicationsIn worse case such as internal hemorrhage whole bloodtransfusion may be carried out [27]
4 Dengue Vaccine Strategies
Despite the existing challenges for an ideal dengue vaccinedevelopment of dengue vaccine candidates has progressedover the last decade and some of these have entered clinicaltrials in both endemic and nonendemic areas A classificationof the current approaches for dengue vaccine development isshown in Figure 5
41 Replicating Viral Vaccines These include live-attenuatedviruses (LAV) that are created by reducing the virulence of apathogen without compromising its viability Current meth-ods of producing live-attenuated viruses for dengue vaccinesinclude attenuation by serial passage in cell lines and targetedmutagenesis and by constructing chimeric vaccine viruses
(i) Advantages robust lasting and broad immunity andlower production cost
(ii) Disadvantages difficulty in attenuation genetic insta-bility possibility of reversion and interference in thecase of multicomponent LAV vaccines
411 Cell Culture Passage Based LAV Development of LAVby serial passage in cell lines was started at Mahidol Univer-sity Bangkok Thailand A tetravalent formulation was madeby attenuating all fourDENV serotypes but the vaccine failedto elicit a balanced immune response despite modulating theviral concentrations [32 33] Increased frequency of adversereactions like fever rash myalgia and retroorbital painprimarily related to theDENV-3 vaccine strain was observedFurther development of these LAV strains was stalled [34]
Another LAV based on passaging in cell culture wasdeveloped by Walter Reed Army Institute of Research(WRAIR) Maryland USA and is being evaluated in clinical
trials in collaboration with GlaxoSmithKline (GSK) Allfour DENV serotypes were attenuated by passaging in pri-mary dog kidney (PDK) cells and a tetravalent formulation(F17Pre) was developed which was found to result in DENV-4 vaccine-induced viremia during phase II clinical trials[35 36] In a separate phase II randomized observer-blindplacebo-controlled trial in 86 healthy flavivirus naıve adultsin USA F17Pre DENVs were rederived and passaged infetus rhesus lung cells to obtain seed viruses of higher purityResultant formulations F17 and F19 containing equivalentamounts of vaccine components except DENV-4 being 10-fold higher in F17 were evaluated An acceptable safetyand immunogenicity profile was observed after 2 doses ofLAV with tetravalent antibody rates of 60 and 67 inparticipants receiving F17 and F19 respectively It was alsoreported that although F19 was formulated to contain 10-foldless DENV-4 it was found to be only fourfold less at the timeof vaccine release The neutralization titers against DENV-4 were found to be comparable at 70 and 46 for F17 andF19 respectively Notably the incidence of DENV-4 vaccine-induced viremia reduced (with only one case in F17 group)probably due to rederivation and passage which attenuatedthe DENV-4 strain further [37] In a similar phase II trialin healthy children and adults in Puerto Rico F17 and F19were evaluated againTheDENV-4 in vitropotency in F19wasfound to be 50-fold less instead of being 10-fold according toformulation design Thus there have been issues related tostorage stability of DENV-4 strain [38]
412 Targeted Mutagenesis Based Live-Attenuated VaccineThis strategy was first successfully explored by the Laboratoryof Infectious Disease at the National Institute of Allergy andInfectious Disease (NIAID) National Institutes of Health(NIH) Maryland USA NIH has established nonexclusivelicense with manufacturers in Brazil (Instituto Butantan)Vietnam (Vabiotech) and India (Serum Institute of Indiaand Panacea Biotech) for its development This vaccinecandidate is amixture of fourDENV strains attenuated by site
Journal of Immunology Research 7
directed mutagenesis to delete 30 nucleotides in the 31015840UTRDENV-1 and DENV-4 attenuated strains were designatedas DEN1Δ30 and DEN4Δ30 respectively [39] DENV-2 andDENV-3 attenuated strains were made by using DEN4Δ30as a backbone and replacing their structural prM and Egenes with those of the corresponding serotype Notablychimerization resulted in overattenuation of rDEN24Δ30and rDEN34Δ30 strainsTheDENV-3 component wasmod-ified variably and rDEN3Δ3031 strain was selected whereadditional 31 nucleotides were deleted from rDEN3Δ30Infectivity of DENV-2 component has been improved intetravalent formulation TV005 by using DENV-2 attenuatedrDEN24Δ30 strain at a 10-fold higher dose (104 pfu) thanother components (103 pfu) tetravalent formulation TV003contains 103 pfu of each of the four components Importantlya single dose of TV005 has been found to be efficacious inproviding sterilizing immunity Additionally TV003TV005are being evaluated in a human challenge model to enable amore stringent assessment of its protective efficacy TV003has been found to protect vaccinees against challenge withDENV-2 attenuated rDEN2Δ30 strain [40 41] Similar evalu-ation of protective efficacy is ongoing for TV005 andDENV-3human challenge experiments are being planned [40] PhaseIII of this vaccine candidate has begun in Brazil [42]
413 Chimeric Dengue Vaccine Chimeric dengue vaccineshave been designed using two approaches (i) with anotherattenuated flavivirus and (ii) with an attenuated DENV strain(intertypic chimera) The vaccine where chimera of DENVhas been made with another flavivirus is the chimeric yellowfever-dengue (CYD) vaccine which is being developed bySanofi Pasteur and licensed under the brand name ldquoDeng-vaxiardquo [4] In this vaccine prM and E genes of the attenuatedyellow fever LAV strain 17D have been replaced with thecorresponding genes from DENV [43] The rationale behindthis design was the fact that humoral response against thestructural proteins of dengue was responsible for protectiveimmunity during natural infection and thus these chimeraswould generate a protective immune response in vaccineesA tetravalent mixture of the four chimeric viruses hasundergone extensive clinical evaluation and has recently beenapproved in Mexico Brazil El Salvador and Philippines[4 44] This vaccine will be discussed in detail in the latersections
An example of intertypic chimera is DENVax developedby Inviragen Inc Fort Collins CO USA DENV-2 strainattenuated by 53 passages in PDK cells (made at MahidolUniversity) has been used as a backbone for generatingchimera prMandE gene of this strainwere replaced by corre-sponding genes fromDENV-1DENV-3 andDENV-4As themutations in the attenuated strain were in the nonstructuralproteins this strain was used as such for DENV-2 componentin the tetravalent formulationThese chimeric viruses showeda temperature-sensitive phenotype reduced replication inmosquito cell lines high degree of genetic stability and lackof neurovirulence in sucklingmice [45]Three tetravalent for-mulations with variable dose of each component were evalu-ated in nonhuman primates It was observed that DENV-2
was the dominating component and its replicative potentialreduced by increasing the DENV-3 andDENV-4 componentThis variation in DENV-2 induced viremia due to the varia-tion in the dose of DENV-3 and DENV-4 components indi-cated viral interference Moreover the neutralizing antibodytiters were found to be significantly low against DENV-4 anddespite this macaques were found to be protected againstDENV-4 challenge [46] A phase I clinical trial of low andhigh doses of the DENVax in healthy subjects in Columbiarevealed that the candidate was safe and immunogenicNotably it corroborated the findings made in nonhuman pri-mate study that neutralizing antibody titers elicited by DEN-Vax are lowest against DENV-4 and highest against DENV-2[47] Further insights into its efficacy will be revealed throughits phase II clinical trial evaluation Meanwhile phase IIIevaluation of this vaccine has now been initiated [48]
42 Nonreplicating Viral Vaccines These vaccine candidatesare not capable of replicating and thus offer the advantage ofconferring immunity without the risk of infection There aremultiple strategies to develop this class of vaccines like DNAvaccines subunit proteins VLPs and so forth
(i) Advantages reduced reactogenicity better suitabilityfor immune-compromised individuals and balancedimmune response in case of tetravalent formulation
(ii) Disadvantages less broad potent and durable im-mune response whichmay result in ADE and requir-ing the use of adjuvants
421 Purified Inactivated Virus (PIV) WRAIR MarylandUSA developed an inactivated monovalent dengue vaccineby formalin treatment It was found to be safe and immuno-genic in mice and Rhesus macaques [49 50] Althoughsuch vaccines would not show viral interference or revertto a pathogenic strain their use as the sole immunizationapproach is limited because of conformational changes invirus by formalin treatment and lack of replication Howeverthis vaccine has been tested as the priming vaccine in a prime-boost immunization strategy with a LAV as the boostervaccine leading to complete protection in macaques [51]Phase I trial evaluating the safety of 25 and 5 120583g of DENV-1component administered on days 0 and 28 in flavivirus naıvepopulation in the USA has been completed [52] Two phase Itrials evaluating tetravalent mixture of the four PIVs (TPIV1 120583g of each of the four PIVs) are being evaluated with alumand two proprietory adjuvants of GSK (AS01E1 and AS03B1)in healthy adults in the USA [53] and in Puerto Rico [54]Healthy adults in the USA are also being recruited in anotherphase I study where TPIValum is being evaluated in prime-boot vaccination with WRAIRGSKrsquos tetravalent LAV [55]
422 Recombinant Subunit Vaccine Recombinant E proteinsof dengue have been expressed in yeast and insect expres-sion systems and have been analyzed for vaccine efficacyin mice and monkeys All these studies have focused onthe DENV E aminoterminal 80 of the molecule knownas the ectodomain Deletion of 20 of the E protein at
8 Journal of Immunology Research
the C-terminal which is a transmembrane region allowsextracellular secretion and easy purification while retainingits antigenicityThe recombinant 80 E proteins also knownas r80E of the four DENV serotypes are being manufac-tured by Hawaii Biotech Inc HI USA and Merck andCo NJ USA Monovalent DEN2 80E was evaluated witha panel of adjuvants in mice and saponin-based adjuvantISCOMATRIXtrade was found to be the most immunogenicimmunogenicity with alum as adjuvant was poor Thiswas followed by evaluation of tetravalent formulation withISCOMATRIX in macaques where titers against DENV-4were found to be the weakest [56] To overcome the lowimmunogenicity of DEN4 80E its dimeric form and doubledose were explored in macaques which led to comparableimprovement in the neutralizing titers against DENV-4Though the titers against DENV-4 improved they were lowerthan the titers against DENV-1 DENV-2 and DENV-3 [57]Based on these results a tetravalent mixture of the four r80Escontaining 10 10 10 and 20120583g of DEN1 DEN2 DEN3and DEN4 80E respectively was further evaluated in flavi-naıve and dengue-primed macaques where it was found togenerate a more balanced immune response against the fourserotypes in 0- 1- and 6-month immunization schedule ascompared to 0 1 and 2 months Moreover two doses (10and 50 120583g) of DEN1 80Ealum administered in flavi-naıveadults on 0 1 and 2 months were found to be safe Howeverit elicited only modest DENV-1 neutralizing titers whichwaned almost completely 26 weeks after the final dose [58]A phase I study examining safety and immunogenicity oftetravalent formulation with and without adjuvant (alum andISCOMATRIX) in healthy adults has been completed [59]
Recombinant antigens based on DENV EDIII have beenproduced by different groups using E coli and yeast expres-sion hosts Recombinant EDIII antigens expressed eitherindependently or fused to different carriers such as maltose-binding protein [60] and the Neisseria meningitides p64kprotein have been shown to generate anti-DENV immuneresponses in mice and nonhuman primates [61ndash63] Thesevaccines candidates are in preclinical phase currently
423 Dengue DNA Vaccine This vaccine consists of a plas-mid vector containing the gene(s) encoding for an antigenwhich on immunization is taken up by antigen presentingcells (APCs) Once the plasmid enters the cell it codes forthe antigen which finally gets associated with MHC classI molecules and gets displayed on the cell surface induc-ing protective cytotoxic immune response Naval MedicalResearch Center (NMRC) USA has developed a DENV-1DNA vaccine candidate (D1ME100) by cloning prM and Egene of DENV-1 serotype into plasmid vector which wasextensively evaluated inmice andmacaques without adjuvant[64] before phase 1 trials in healthy adults AlthoughDENV-1DNA vaccine was found to be well tolerated the neutralizingantibody titers and the number of responders were foundto be low [64 65] Thus to enhance its immunogenicitya lipid-based adjuvant Vaxfectin was explored Tetravalentdengue DNA vaccine (TVDV) was evaluated for immuno-genicity with and without Vaxfectin in macaques It wasobserved that Vaxfectin resulted in higher and more stable
(evaluated till 6 months after final boost) titers The averageneutralization titers with TVDVVaxfectin against DENV-1DENV-2 DENV-3 and DENV-4 a month after final boostwere approximately 200 270 170 and 70 respectively Sixmonths after the final boost the titers against DENV-2 andDENV-3 reduced while those against DENV-1 and DENV-4increased marginally In the group without Vaxfectin titersagainst DENV-2 only were detectable 6 months after the finalboost Moreover Vaxfectin allowed better protection fromviremia against DENV-2 challenge [66] After establishingnontoxicity of TVDVVaxfectin in New Zealand white rab-bits [67] a phase I trial was initiated in 2011 in USA [68]
424 Replication-Defective Virus Vectored Vaccines In thisapproach a virus is used as a vector to carry antigenic genesthat are capable of eliciting neutralizing antibody responseSome examples of viral vectors are adenovirus vectors Ven-ezuelan equine encephalitis virus vector and attenuatedmeasles virus [69ndash71] An example of virus vectored denguevaccine is cAdVax It consists of bivalent constructs express-ing prM and E proteins from two dengue serotypes each(DENV-1 and DENV-3 together in one and DENV-2 andDENV-4 in another construct) Study in NHPs showedproduction of neutralizing antibodies to respective DENVserotypes [72 73] Therefore a tetravalent formulation (cAd-Vax-DenTV) was prepared bymixing the bivalent constructswhich showed protection against all serotypes on DENVchallenge in Rhesus macaques [69]
425 Virus Like Particle (VLP) Vaccines The prM and Eproteins of DENVs coexpressed in heterologous hosts havebeen shown to coassemble into VLPs Thus a vaccine basedon physical mixtures of four monovalent DENV VLPs canbe developed to have a tetravalent formulation [74] Fromthe perspective of using VLPs for vaccine purpose the yeastsystemmay bemore suitable as it has the potential for higheryields and can glycosylate the antigens Recent work indicatesthat yeast expressed-DENV E ectodomain forms VLPs in theabsence of prM [75] Another approach is based on displayingthe DENV EDIII on VLPs formed by hepatitis B virus coreantigens [27]
5 Dengvaxia
Dengue vaccine candidates which have reached clinical trialsare given in Table 2 It is worthwhile to discuss the front-runner CYD vaccine developed by Sanofi Pasteur whichhas recently been approved as Dengvaxia in Mexico BrazilEl Salvador and Philippines [4] Dengvaxia is a tetravalentdengue chimeric live-attenuated virus vaccine based onlicensed yellow fever vaccine 17D It was constructed byreplacement of structural genes of live-attenuated yellowfever virus vaccine 17D with structural genes from eachDENV serotype [76]
51 Preclinical Data The immunogenicity of various tetrava-lent formulations of the chimeric viruses was evaluated inmacaques which revealed immunodominance of serotype
Journal of Immunology Research 9
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
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[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
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[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
Figure 1 Genome organization and membrane topology of dengue virus The viral RNA is translated as a single polyprotein consistingof structural (light brown-C prM and E) and nonstructural (dark brown-NS1 2A 2B 3 4A 4B and 5) protein components Symbols CprM E NS and PM denote capsid protein precursor membrane protein envelope protein nonstructural proteins and plasma membranerespectivelyThis single polyprotein then gets processed by viral (green arrow) and host (black arrow) proteasesThe structural proteins (prMand E) remain anchored on the luminal side of the ER membrane The C protein is anchored on the cytoplasmic side of ER membrane prMis later cleaved by furin (red arrow) in the TGN into the pr peptide and M protein The NS proteins are mainly processed by NS2B-NS3(viral protease) in the cytoplasm NS2A2B and NS4A4B are transmembrane proteins and thus stay anchored in the ER The approximatemolecular weight (in kDa) of each protein has been indicated in braces
complex and Ae niveus have been found to play a roleas secondary vectors [8] However Ae niveus is consideredonly as a sylvatic vector The life cycle of Aedes mosquitodepending upon the extent of feeding lasts for 8ndash10 days atroom temperature It consists of two phases aquatic (larvaepupae) and terrestrial (eggs adults) phase Presently Aealbopictus has become an increasingly important vector as itcan easily adapt to new environments including temperateregions Its spread to Ae Aegypti free countries has createdopportunities for dengue viruses to enter new locations andcause disease [10] However it is still a minor contributor tohuman dengue infections
2 Dengue Virus
21 Genomic Structure The viral genome consists of a posi-tive sense RNA of sim11 kb This RNA is translated into a singlepolyprotein which encodes for three structural proteinsnamely capsid (C) premembrane (prM) envelope (E) and 7nonstructural proteins (NS1NS2ANS2BNS3NS4ANS4Band NS5) (Figure 1)
It consists of a single open reading frame and two non-coding regions (NCRs) at the 51015840 and 31015840 ends It is expressedas a single polyprotein precursor which is coposttransla-tionally cleaved by viral and host proteases (Figure 1) The51015840 and 31015840 NCRs contain secondary structures and conservedsequences which are involved in regulation of viral repli-cation The 51015840UTR (sim100 nucleotides) has a type I methy-lated cap structure (m7G51015840ppp51015840A) but the 31015840UTR (sim450nucleotides) lacks a terminal polyadenylate tail Proteinsynthesis occurs in the cytoplasm on the Rough Endoplasmic
Reticulum (RER) and the structural proteins get anchored tothe ER on the luminal side where assembly and maturationof virion occur (Figure 1) [2 11] Major functions of all theproteins are summarized in Table 1 [11 12]
22 Structure of Virion and Envelope Protein A three-dimen-sional image reconstruction of mature dengue virus showsthat it is sim50 nm in diameter and consists of an outer proteinshell (E and M) a lipid bilayer and a less characterizednucleocapsid core (C and RNA genome) Dengue virusexhibits different surface structures during itsmaturation andinfection and these conformational changes are attributed tothe inherent flexibility of the envelope protein E protein ismade up of three domains namely EDI (red) EDII (yellow)and EDIII (blue) and transitions between its oligomericstates are supported by the hinge motion that occurs betweenEDI-EDII and EDI-EDIII The immature virus particle hasa spiky appearance with 60 trimeric surface spikes eachconsisting of three prM-E heterodimers (Figure 2) The prpeptides are cleaved in the TGN by furin which leads torearrangement of the E proteins into 90 homodimers of E thatlie flat against the viral surface giving a smooth appearance acharacteristic ofmature virus In an E dimer the Emonomersare arranged face to face with their long directions beingantiparallel to each other M remains anchored to the lipidbilayer below the E protein shell (Figure 2)
When mature virus infects a new cell through receptor-mediated endocytosis the E protein molecules shift to atrimeric conformation in the acidic compartment of an endo-some protrude from the virus surface causing membranefusion and facilitate viral RNA release into the cytoplasm(Figure 2) [2 13 14]
Journal of Immunology Research 3
Table 1 Functions of DENV proteins
Protein FunctionStructural
Capsid (C) Binds and stabilizes viral RNA
Premembranemembrane (prMM)(i) Pr peptide functions as cap that protects the fusion peptide on E thus preventingpremature fusion(ii) M forms ion channel
Envelope (E) (i) Recognition and binding to the host cell(ii) Involved in uncoating of virus by enabling fusion of viral and endosomal membranes
Nonstructural (NS)NS1 (i) Viral RNA replication
(ii) Viral defense through inhibition of complement activationNS2A Viral replication and assemblyNS2B NS3 protease cofactorNS3 (i) Serine protease-cleaves viral polyprotein
(ii) RNA helicase and RTPaseNTPase-viral RNA replication(iii) Induction of apoptosis in infected cells
NS4A Induces membrane alterations and autophagy to enhance virus replicationNS4B (i) Interacts with NS3-viral replication
(ii) Blocks IFN-120572120573-induced signal transduction and helps virus to escape hostrsquos innateimmune response
Figure 2 Organization of E protein on dengue virus surface duringits life cycle The E protein is colored as follows EDI (red) EDII(yellow) EDIII (blue) and the FL (green) prM and M proteinare colored as cyan (a) Immature virus contains 60 trimericspikes of E and prM heterodimer (b) Mature virus contains 90homodimers of E protein (c) These homodimers then furtherundergo reorganization to form fusion active E homotrimers inwhich fusion loop is exposed M protein is not shown in the fusiontrimer for simplicity E C M FL and prM denote envelope capsidmembrane fusion loop and precursor membrane respectively
The E protein is the major exposed antigen of the denguevirion antibodies against which provide immunity duringnatural infectionThe E proteins of the four DENV serotypeshave 60ndash70 amino acid similarity and are glycosylated atAsn-67 (unique to dengue) and Asn-153 These residues havebeen found to play important roles in the receptor attachmentand viral entry into the cell The E protein consists of atransmembrane region and an ectodomain which is dividedinto three structuralfunctional domains [15]
(i) EDI (envelope domain I) central region contains 8-stranded 120573-barrel and organizes the structure
(ii) EDII (envelope domain II) is a dimerization domainand contains 12 120573 strands 2 120572 helices and a highlyconserved fusion loop
(iii) EDIII (envelope domain III) contains immunoglob-ulin like domain with 10 120573 strands and is involved inreceptor binding
Apart from the primary difference in their structure the threedomains of the ectodomain differ in their immunogenicity asillustrated schematically in Figure 3 The EDIII of each of thefour serotypes (circled and colored in red green blue andblack for DENV-1 DENV-2 DENV-3 and DENV-4 resp)elicits strongly neutralizing antibodies which are largelyserotype-specific [16] Strongly neutralizing serotype-specificantibodies have been largely found to be elicited againstthe EDIII hinge region [17] complex quaternary epitopesdisplayed on the E protein dimer and thewhole virion [18 19]
4 Journal of Immunology Research
Figure 3 DENV-1 DENV-2 DENV-3 and DENV-4 with EDIII(circled) in red green blue and black respectively EDIIIs elicitstrongly neutralizing serotype-specific antibodies (antibodies inbold) Owing to homology especially in domains (EDIII and prM)in yellow cross-reactive weakly neutralizing antibodies (unboldantibodies) and nonneutralizing enhancing antibodies (dashedantibodies) are elicited in bulk
It has been reported that it is serotype-specific neutralizingantibodies and not cross-reactive neutralizing antibodiesthat confer protection against infection [20] It has beenreported that bulk of the immune response is elicitedagainst the cross-reactive domains of EDI EDII and prM(yellow domain in the virus image) [16 21] Anti-EDIIIantibodies (Figure 3 unbold antibodies) are largely het-erotypic weaklynonneutralizing while prM antibodies (Fig-ure 3 dashed antibodies) are largely nonneutralizing andcross-reactive It is believed that the virus utilizes suchweaknonneutralizing cross-reactive antibodies in gainingaccess into the host cell via Fc receptor as an alternativepathway during a secondary infection with a heterologousserotype leading to enhancement of infection [16 20 21]This phenomenon is known as antibody-dependent enhance-ment (ADE)
Thus it can be inferred that strongly neutralizing anti-bodies prevent ADE caused by a larger population of weaklynonneutralizing enhancing antibodies A vaccine candidatecapable of a larger population of strongly neutralizing anti-bodies could probably be an ideal vaccine showcasing strongprotection without ADE This can probably be achieved bydesigning the vaccine candidate and not by default strategy
23 Cellular Targets and Receptor Interaction The currentmodel of flavivirus cell entry suggests the use of two func-tionally different sets of molecules attachment factors thathelp the virus to concentrate on the cell surface and primaryreceptor(s) which help directing the virion to the endocyticpathway (Figure 4) [22]
During natural dengue infection in humans themosquito delivers virus in skin epithelium where it infectsand replicates in the cells of mononuclear lineage like mono-cytes dendritic cells macrophages and Langerhans cells[23 24] These infected cells carry the virus to lymph nodeswhere it replicates resulting in viremia which is followedby systemic infection of liver lungs and spleen Howeverin mosquitoes the primary target of DENV infection is theepithelium of the midgut where it first replicates and then
[25 26] spreads to and replicates in salivary glands fromwhere the infection is transmitted through saliva to the nextvertebrate host during the blood meal
3 Dengue Disease
31 Classification The spectrum of clinical illness may rangefrom asymptomatic disease to a broad range of syndromeswith severe clinical manifestations Symptomatic infectionmay range frommild debilitating DF to life threatening DHFand DSS due to plasma leakage in DHF patients Thesethree conditions likely represent progressively severe stagesof a continuous dengue disease spectrum [27] They arebased on traditional WHO classification case definitions andcontinue to be recognized in many regions of the worlddespite the introduction of a new classification system Thenew classification based on a single parameter [5] allowsbetter case capture [28] but is not compatible with restrictedhealth care facilities in endemic regions especially duringoutbreaks [29]
32 DF DF is a self-limiting fever lasting usually for 5ndash7days It is sometimes debilitating during the acute illnessstage The clinical features of DF vary according to the ageof the patient The infants and young children may haveundifferentiated febrile sickness with maculopapular rashThe older children and adults may have mild febrile syn-drome or severe disease with high fever (usually bipha-sic) severe headache retroorbital pain myalgia arthralgianausea vomiting and petechiae Leukopenia and thrombo-cytopenia are usually observed in all ages In some casesDF may accompany bleeding complication such as gingivalbleeding epistaxis gastrointestinal bleeding haematuriaand menorrhagia (in case of women) [27]
33 DHFDSS DHF is characterized by symptoms of DFalong with thrombocytopenia hemorrhagic manifestationsand plasma leakage A positive tourniquet test may be sug-gestive of DHF however this is being debated now due to itslow sensitivityspecificity Plasma leakage determines diseaseseverity in DHF It is also the most important differencebetween DHF and DF Depending on disease severity andclinical manifestations DHF is divided into four grades Ito IV with grade IV being the most severe Several patientsalso have fine petechiae scattered on the extremities axillaeface and soft palate usually seen in the febrile periodThe critical phase is usually reached at the end of febrileillness marked by rapid decrease in temperature and oftenaccompanied by circulatory disturbances including plasmaleakage hemoconcentration and thrombocytopenia [27]
In severe cases with critical plasma loss DSS ensuesand may be life threatening if not treated properly DSS ischaracterized by a rapid weak pulse with narrowing pulsepressure (lt20mmofHg) cold clammy skin and restlessnessThe patient may die within 12ndash24 h of going into shock orrecover rapidly with volume replacement therapy
34 Primary and Secondary Dengue Infection The first expo-sure of an individual to any of the four dengue virus serotypes
Journal of Immunology Research 5
Mature dengue virion
GRP78Mannose receptor
Heparan sulfate
TAMTIM
HSP 7090NKp44DC-SIGN
Clathrin coated pit Internalisation via endocytic pathway
(A) (B)
PM
Outside
Cytoplasm
Figure 4 Schematic representation of the dengue virus entry process The dengue virus makes use of membrane receptors and attachmentfactors on the cell plasma membrane (PM) to find its way to the cytoplasm The mature virion either gets attached directly to a cellularmembrane receptor (a) or uses several attachment factors (b) to finally trigger the endocytic clathrin dependent pathway The endocyticvesicle becomes a late endosome where acidification triggers conformational changes on the E protein dimers to become fusogenic trimersFinally pores are formed and the genome of the virus is released into the cytoplasm
is known as primary dengue infection It maymay not resultin symptomatic infection In primary infection high titersof immunoglobulin M (IgM) and immunoglobulin G (IgG)antibodies appear in 3ndash5 and 6ndash10 days respectively afterthe onset of infection The presence of IgM is transientdisappearing in 2-3 months after the onset of illness whereasIgG persists for life [30] Hence primary infection with aparticular serotype provides life-long immunity against thatserotype But it does not provide continued cross-protectiveimmunity against the remaining serotypes
A secondary infection with a previously unencounteredDENV serotype usually results in classical DF However 2-3of secondary infection cases develop intoDHFwhichmayprogress to DSS and death During a second infection with adifferent serotype the presence of low amounts of heterotypicantibodies (which form complexes with DENVs) promotesthe access of the virus to monocytes via Fc receptors leadingto an increase in viral load and severity of the disease Thisphenomenon is known as ADE The major players of thisphenomenon are cross-reactive antibodies elicited againstthe fusion loop and prM which are found to be weaklyneutralizing leading to enhancement of infection at lowconcentrations [16 21] Although ADE has been found toresult in disease severity all the severe cases are not associatedwith secondary infection nor do all the cases of secondaryinfection progress to DHFDSS [2] In addition to humoralimmunity cross-reactive memory T cells could also playa role in either providing protective immunity or causingimmunopathology [31]
35 Diagnosis and Clinical Management Dengue infectionis usually confirmed by identification of viral genomic RNAantigens or the antibodies it elicits Antigen detection testsbased on NS1 detection have been designed to detect thedengue viral NS1 proteinwhich gets released from the dengueinfected cells and appears early in the bloodstream A 3-in-1test for simultaneous detection of NS1 IgM and IgG is nowavailable ELISA-based serological tests are easy to performand are cost-effective for dengue detection
Up to date there is no antiviral drug available fordengue Treatment is usually based on symptoms and isperformed through medical support For uncomplicatedcases of dengue fever the treatment prescribed is bed restoral rehydration and paracetamol as an antipyretic andanalgesic Patientrsquos health is monitored through variousblood tests from fever day 3 onwards till the conditionimproves Clinical signs that signal progression to seriousdisease include cold limb extremities low pulse low urineoutput signs of mucosal bleeding and abdominal pain DHFis indicated by a rising hematocrit (ge20) and a fallingplatelet count (gt100000mm3) If any of these signs aredetected immediate hospitalization is necessary Treatmentfor DHF patients is based on intravenous fluid therapy tomaintain effective circulation during plasma leakage pluscareful clinical monitoring of hematocrit platelet countpulse rate and blood pressure temperature urine outputfluid administered and other signs of shock Patients usuallyrecover within 12ndash48 h of fluid therapy Treatment for DSSpatients mainly consists of immediate fluid therapy with
Figure 5 Classification of dengue vaccine candidates
colloids and extensive monitoring of any complicationsIn worse case such as internal hemorrhage whole bloodtransfusion may be carried out [27]
4 Dengue Vaccine Strategies
Despite the existing challenges for an ideal dengue vaccinedevelopment of dengue vaccine candidates has progressedover the last decade and some of these have entered clinicaltrials in both endemic and nonendemic areas A classificationof the current approaches for dengue vaccine development isshown in Figure 5
41 Replicating Viral Vaccines These include live-attenuatedviruses (LAV) that are created by reducing the virulence of apathogen without compromising its viability Current meth-ods of producing live-attenuated viruses for dengue vaccinesinclude attenuation by serial passage in cell lines and targetedmutagenesis and by constructing chimeric vaccine viruses
(i) Advantages robust lasting and broad immunity andlower production cost
(ii) Disadvantages difficulty in attenuation genetic insta-bility possibility of reversion and interference in thecase of multicomponent LAV vaccines
411 Cell Culture Passage Based LAV Development of LAVby serial passage in cell lines was started at Mahidol Univer-sity Bangkok Thailand A tetravalent formulation was madeby attenuating all fourDENV serotypes but the vaccine failedto elicit a balanced immune response despite modulating theviral concentrations [32 33] Increased frequency of adversereactions like fever rash myalgia and retroorbital painprimarily related to theDENV-3 vaccine strain was observedFurther development of these LAV strains was stalled [34]
Another LAV based on passaging in cell culture wasdeveloped by Walter Reed Army Institute of Research(WRAIR) Maryland USA and is being evaluated in clinical
trials in collaboration with GlaxoSmithKline (GSK) Allfour DENV serotypes were attenuated by passaging in pri-mary dog kidney (PDK) cells and a tetravalent formulation(F17Pre) was developed which was found to result in DENV-4 vaccine-induced viremia during phase II clinical trials[35 36] In a separate phase II randomized observer-blindplacebo-controlled trial in 86 healthy flavivirus naıve adultsin USA F17Pre DENVs were rederived and passaged infetus rhesus lung cells to obtain seed viruses of higher purityResultant formulations F17 and F19 containing equivalentamounts of vaccine components except DENV-4 being 10-fold higher in F17 were evaluated An acceptable safetyand immunogenicity profile was observed after 2 doses ofLAV with tetravalent antibody rates of 60 and 67 inparticipants receiving F17 and F19 respectively It was alsoreported that although F19 was formulated to contain 10-foldless DENV-4 it was found to be only fourfold less at the timeof vaccine release The neutralization titers against DENV-4 were found to be comparable at 70 and 46 for F17 andF19 respectively Notably the incidence of DENV-4 vaccine-induced viremia reduced (with only one case in F17 group)probably due to rederivation and passage which attenuatedthe DENV-4 strain further [37] In a similar phase II trialin healthy children and adults in Puerto Rico F17 and F19were evaluated againTheDENV-4 in vitropotency in F19wasfound to be 50-fold less instead of being 10-fold according toformulation design Thus there have been issues related tostorage stability of DENV-4 strain [38]
412 Targeted Mutagenesis Based Live-Attenuated VaccineThis strategy was first successfully explored by the Laboratoryof Infectious Disease at the National Institute of Allergy andInfectious Disease (NIAID) National Institutes of Health(NIH) Maryland USA NIH has established nonexclusivelicense with manufacturers in Brazil (Instituto Butantan)Vietnam (Vabiotech) and India (Serum Institute of Indiaand Panacea Biotech) for its development This vaccinecandidate is amixture of fourDENV strains attenuated by site
Journal of Immunology Research 7
directed mutagenesis to delete 30 nucleotides in the 31015840UTRDENV-1 and DENV-4 attenuated strains were designatedas DEN1Δ30 and DEN4Δ30 respectively [39] DENV-2 andDENV-3 attenuated strains were made by using DEN4Δ30as a backbone and replacing their structural prM and Egenes with those of the corresponding serotype Notablychimerization resulted in overattenuation of rDEN24Δ30and rDEN34Δ30 strainsTheDENV-3 component wasmod-ified variably and rDEN3Δ3031 strain was selected whereadditional 31 nucleotides were deleted from rDEN3Δ30Infectivity of DENV-2 component has been improved intetravalent formulation TV005 by using DENV-2 attenuatedrDEN24Δ30 strain at a 10-fold higher dose (104 pfu) thanother components (103 pfu) tetravalent formulation TV003contains 103 pfu of each of the four components Importantlya single dose of TV005 has been found to be efficacious inproviding sterilizing immunity Additionally TV003TV005are being evaluated in a human challenge model to enable amore stringent assessment of its protective efficacy TV003has been found to protect vaccinees against challenge withDENV-2 attenuated rDEN2Δ30 strain [40 41] Similar evalu-ation of protective efficacy is ongoing for TV005 andDENV-3human challenge experiments are being planned [40] PhaseIII of this vaccine candidate has begun in Brazil [42]
413 Chimeric Dengue Vaccine Chimeric dengue vaccineshave been designed using two approaches (i) with anotherattenuated flavivirus and (ii) with an attenuated DENV strain(intertypic chimera) The vaccine where chimera of DENVhas been made with another flavivirus is the chimeric yellowfever-dengue (CYD) vaccine which is being developed bySanofi Pasteur and licensed under the brand name ldquoDeng-vaxiardquo [4] In this vaccine prM and E genes of the attenuatedyellow fever LAV strain 17D have been replaced with thecorresponding genes from DENV [43] The rationale behindthis design was the fact that humoral response against thestructural proteins of dengue was responsible for protectiveimmunity during natural infection and thus these chimeraswould generate a protective immune response in vaccineesA tetravalent mixture of the four chimeric viruses hasundergone extensive clinical evaluation and has recently beenapproved in Mexico Brazil El Salvador and Philippines[4 44] This vaccine will be discussed in detail in the latersections
An example of intertypic chimera is DENVax developedby Inviragen Inc Fort Collins CO USA DENV-2 strainattenuated by 53 passages in PDK cells (made at MahidolUniversity) has been used as a backbone for generatingchimera prMandE gene of this strainwere replaced by corre-sponding genes fromDENV-1DENV-3 andDENV-4As themutations in the attenuated strain were in the nonstructuralproteins this strain was used as such for DENV-2 componentin the tetravalent formulationThese chimeric viruses showeda temperature-sensitive phenotype reduced replication inmosquito cell lines high degree of genetic stability and lackof neurovirulence in sucklingmice [45]Three tetravalent for-mulations with variable dose of each component were evalu-ated in nonhuman primates It was observed that DENV-2
was the dominating component and its replicative potentialreduced by increasing the DENV-3 andDENV-4 componentThis variation in DENV-2 induced viremia due to the varia-tion in the dose of DENV-3 and DENV-4 components indi-cated viral interference Moreover the neutralizing antibodytiters were found to be significantly low against DENV-4 anddespite this macaques were found to be protected againstDENV-4 challenge [46] A phase I clinical trial of low andhigh doses of the DENVax in healthy subjects in Columbiarevealed that the candidate was safe and immunogenicNotably it corroborated the findings made in nonhuman pri-mate study that neutralizing antibody titers elicited by DEN-Vax are lowest against DENV-4 and highest against DENV-2[47] Further insights into its efficacy will be revealed throughits phase II clinical trial evaluation Meanwhile phase IIIevaluation of this vaccine has now been initiated [48]
42 Nonreplicating Viral Vaccines These vaccine candidatesare not capable of replicating and thus offer the advantage ofconferring immunity without the risk of infection There aremultiple strategies to develop this class of vaccines like DNAvaccines subunit proteins VLPs and so forth
(i) Advantages reduced reactogenicity better suitabilityfor immune-compromised individuals and balancedimmune response in case of tetravalent formulation
(ii) Disadvantages less broad potent and durable im-mune response whichmay result in ADE and requir-ing the use of adjuvants
421 Purified Inactivated Virus (PIV) WRAIR MarylandUSA developed an inactivated monovalent dengue vaccineby formalin treatment It was found to be safe and immuno-genic in mice and Rhesus macaques [49 50] Althoughsuch vaccines would not show viral interference or revertto a pathogenic strain their use as the sole immunizationapproach is limited because of conformational changes invirus by formalin treatment and lack of replication Howeverthis vaccine has been tested as the priming vaccine in a prime-boost immunization strategy with a LAV as the boostervaccine leading to complete protection in macaques [51]Phase I trial evaluating the safety of 25 and 5 120583g of DENV-1component administered on days 0 and 28 in flavivirus naıvepopulation in the USA has been completed [52] Two phase Itrials evaluating tetravalent mixture of the four PIVs (TPIV1 120583g of each of the four PIVs) are being evaluated with alumand two proprietory adjuvants of GSK (AS01E1 and AS03B1)in healthy adults in the USA [53] and in Puerto Rico [54]Healthy adults in the USA are also being recruited in anotherphase I study where TPIValum is being evaluated in prime-boot vaccination with WRAIRGSKrsquos tetravalent LAV [55]
422 Recombinant Subunit Vaccine Recombinant E proteinsof dengue have been expressed in yeast and insect expres-sion systems and have been analyzed for vaccine efficacyin mice and monkeys All these studies have focused onthe DENV E aminoterminal 80 of the molecule knownas the ectodomain Deletion of 20 of the E protein at
8 Journal of Immunology Research
the C-terminal which is a transmembrane region allowsextracellular secretion and easy purification while retainingits antigenicityThe recombinant 80 E proteins also knownas r80E of the four DENV serotypes are being manufac-tured by Hawaii Biotech Inc HI USA and Merck andCo NJ USA Monovalent DEN2 80E was evaluated witha panel of adjuvants in mice and saponin-based adjuvantISCOMATRIXtrade was found to be the most immunogenicimmunogenicity with alum as adjuvant was poor Thiswas followed by evaluation of tetravalent formulation withISCOMATRIX in macaques where titers against DENV-4were found to be the weakest [56] To overcome the lowimmunogenicity of DEN4 80E its dimeric form and doubledose were explored in macaques which led to comparableimprovement in the neutralizing titers against DENV-4Though the titers against DENV-4 improved they were lowerthan the titers against DENV-1 DENV-2 and DENV-3 [57]Based on these results a tetravalent mixture of the four r80Escontaining 10 10 10 and 20120583g of DEN1 DEN2 DEN3and DEN4 80E respectively was further evaluated in flavi-naıve and dengue-primed macaques where it was found togenerate a more balanced immune response against the fourserotypes in 0- 1- and 6-month immunization schedule ascompared to 0 1 and 2 months Moreover two doses (10and 50 120583g) of DEN1 80Ealum administered in flavi-naıveadults on 0 1 and 2 months were found to be safe Howeverit elicited only modest DENV-1 neutralizing titers whichwaned almost completely 26 weeks after the final dose [58]A phase I study examining safety and immunogenicity oftetravalent formulation with and without adjuvant (alum andISCOMATRIX) in healthy adults has been completed [59]
Recombinant antigens based on DENV EDIII have beenproduced by different groups using E coli and yeast expres-sion hosts Recombinant EDIII antigens expressed eitherindependently or fused to different carriers such as maltose-binding protein [60] and the Neisseria meningitides p64kprotein have been shown to generate anti-DENV immuneresponses in mice and nonhuman primates [61ndash63] Thesevaccines candidates are in preclinical phase currently
423 Dengue DNA Vaccine This vaccine consists of a plas-mid vector containing the gene(s) encoding for an antigenwhich on immunization is taken up by antigen presentingcells (APCs) Once the plasmid enters the cell it codes forthe antigen which finally gets associated with MHC classI molecules and gets displayed on the cell surface induc-ing protective cytotoxic immune response Naval MedicalResearch Center (NMRC) USA has developed a DENV-1DNA vaccine candidate (D1ME100) by cloning prM and Egene of DENV-1 serotype into plasmid vector which wasextensively evaluated inmice andmacaques without adjuvant[64] before phase 1 trials in healthy adults AlthoughDENV-1DNA vaccine was found to be well tolerated the neutralizingantibody titers and the number of responders were foundto be low [64 65] Thus to enhance its immunogenicitya lipid-based adjuvant Vaxfectin was explored Tetravalentdengue DNA vaccine (TVDV) was evaluated for immuno-genicity with and without Vaxfectin in macaques It wasobserved that Vaxfectin resulted in higher and more stable
(evaluated till 6 months after final boost) titers The averageneutralization titers with TVDVVaxfectin against DENV-1DENV-2 DENV-3 and DENV-4 a month after final boostwere approximately 200 270 170 and 70 respectively Sixmonths after the final boost the titers against DENV-2 andDENV-3 reduced while those against DENV-1 and DENV-4increased marginally In the group without Vaxfectin titersagainst DENV-2 only were detectable 6 months after the finalboost Moreover Vaxfectin allowed better protection fromviremia against DENV-2 challenge [66] After establishingnontoxicity of TVDVVaxfectin in New Zealand white rab-bits [67] a phase I trial was initiated in 2011 in USA [68]
424 Replication-Defective Virus Vectored Vaccines In thisapproach a virus is used as a vector to carry antigenic genesthat are capable of eliciting neutralizing antibody responseSome examples of viral vectors are adenovirus vectors Ven-ezuelan equine encephalitis virus vector and attenuatedmeasles virus [69ndash71] An example of virus vectored denguevaccine is cAdVax It consists of bivalent constructs express-ing prM and E proteins from two dengue serotypes each(DENV-1 and DENV-3 together in one and DENV-2 andDENV-4 in another construct) Study in NHPs showedproduction of neutralizing antibodies to respective DENVserotypes [72 73] Therefore a tetravalent formulation (cAd-Vax-DenTV) was prepared bymixing the bivalent constructswhich showed protection against all serotypes on DENVchallenge in Rhesus macaques [69]
425 Virus Like Particle (VLP) Vaccines The prM and Eproteins of DENVs coexpressed in heterologous hosts havebeen shown to coassemble into VLPs Thus a vaccine basedon physical mixtures of four monovalent DENV VLPs canbe developed to have a tetravalent formulation [74] Fromthe perspective of using VLPs for vaccine purpose the yeastsystemmay bemore suitable as it has the potential for higheryields and can glycosylate the antigens Recent work indicatesthat yeast expressed-DENV E ectodomain forms VLPs in theabsence of prM [75] Another approach is based on displayingthe DENV EDIII on VLPs formed by hepatitis B virus coreantigens [27]
5 Dengvaxia
Dengue vaccine candidates which have reached clinical trialsare given in Table 2 It is worthwhile to discuss the front-runner CYD vaccine developed by Sanofi Pasteur whichhas recently been approved as Dengvaxia in Mexico BrazilEl Salvador and Philippines [4] Dengvaxia is a tetravalentdengue chimeric live-attenuated virus vaccine based onlicensed yellow fever vaccine 17D It was constructed byreplacement of structural genes of live-attenuated yellowfever virus vaccine 17D with structural genes from eachDENV serotype [76]
51 Preclinical Data The immunogenicity of various tetrava-lent formulations of the chimeric viruses was evaluated inmacaques which revealed immunodominance of serotype
Journal of Immunology Research 9
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
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[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
Premembranemembrane (prMM)(i) Pr peptide functions as cap that protects the fusion peptide on E thus preventingpremature fusion(ii) M forms ion channel
Envelope (E) (i) Recognition and binding to the host cell(ii) Involved in uncoating of virus by enabling fusion of viral and endosomal membranes
Nonstructural (NS)NS1 (i) Viral RNA replication
(ii) Viral defense through inhibition of complement activationNS2A Viral replication and assemblyNS2B NS3 protease cofactorNS3 (i) Serine protease-cleaves viral polyprotein
(ii) RNA helicase and RTPaseNTPase-viral RNA replication(iii) Induction of apoptosis in infected cells
NS4A Induces membrane alterations and autophagy to enhance virus replicationNS4B (i) Interacts with NS3-viral replication
(ii) Blocks IFN-120572120573-induced signal transduction and helps virus to escape hostrsquos innateimmune response
Figure 2 Organization of E protein on dengue virus surface duringits life cycle The E protein is colored as follows EDI (red) EDII(yellow) EDIII (blue) and the FL (green) prM and M proteinare colored as cyan (a) Immature virus contains 60 trimericspikes of E and prM heterodimer (b) Mature virus contains 90homodimers of E protein (c) These homodimers then furtherundergo reorganization to form fusion active E homotrimers inwhich fusion loop is exposed M protein is not shown in the fusiontrimer for simplicity E C M FL and prM denote envelope capsidmembrane fusion loop and precursor membrane respectively
The E protein is the major exposed antigen of the denguevirion antibodies against which provide immunity duringnatural infectionThe E proteins of the four DENV serotypeshave 60ndash70 amino acid similarity and are glycosylated atAsn-67 (unique to dengue) and Asn-153 These residues havebeen found to play important roles in the receptor attachmentand viral entry into the cell The E protein consists of atransmembrane region and an ectodomain which is dividedinto three structuralfunctional domains [15]
(i) EDI (envelope domain I) central region contains 8-stranded 120573-barrel and organizes the structure
(ii) EDII (envelope domain II) is a dimerization domainand contains 12 120573 strands 2 120572 helices and a highlyconserved fusion loop
(iii) EDIII (envelope domain III) contains immunoglob-ulin like domain with 10 120573 strands and is involved inreceptor binding
Apart from the primary difference in their structure the threedomains of the ectodomain differ in their immunogenicity asillustrated schematically in Figure 3 The EDIII of each of thefour serotypes (circled and colored in red green blue andblack for DENV-1 DENV-2 DENV-3 and DENV-4 resp)elicits strongly neutralizing antibodies which are largelyserotype-specific [16] Strongly neutralizing serotype-specificantibodies have been largely found to be elicited againstthe EDIII hinge region [17] complex quaternary epitopesdisplayed on the E protein dimer and thewhole virion [18 19]
4 Journal of Immunology Research
Figure 3 DENV-1 DENV-2 DENV-3 and DENV-4 with EDIII(circled) in red green blue and black respectively EDIIIs elicitstrongly neutralizing serotype-specific antibodies (antibodies inbold) Owing to homology especially in domains (EDIII and prM)in yellow cross-reactive weakly neutralizing antibodies (unboldantibodies) and nonneutralizing enhancing antibodies (dashedantibodies) are elicited in bulk
It has been reported that it is serotype-specific neutralizingantibodies and not cross-reactive neutralizing antibodiesthat confer protection against infection [20] It has beenreported that bulk of the immune response is elicitedagainst the cross-reactive domains of EDI EDII and prM(yellow domain in the virus image) [16 21] Anti-EDIIIantibodies (Figure 3 unbold antibodies) are largely het-erotypic weaklynonneutralizing while prM antibodies (Fig-ure 3 dashed antibodies) are largely nonneutralizing andcross-reactive It is believed that the virus utilizes suchweaknonneutralizing cross-reactive antibodies in gainingaccess into the host cell via Fc receptor as an alternativepathway during a secondary infection with a heterologousserotype leading to enhancement of infection [16 20 21]This phenomenon is known as antibody-dependent enhance-ment (ADE)
Thus it can be inferred that strongly neutralizing anti-bodies prevent ADE caused by a larger population of weaklynonneutralizing enhancing antibodies A vaccine candidatecapable of a larger population of strongly neutralizing anti-bodies could probably be an ideal vaccine showcasing strongprotection without ADE This can probably be achieved bydesigning the vaccine candidate and not by default strategy
23 Cellular Targets and Receptor Interaction The currentmodel of flavivirus cell entry suggests the use of two func-tionally different sets of molecules attachment factors thathelp the virus to concentrate on the cell surface and primaryreceptor(s) which help directing the virion to the endocyticpathway (Figure 4) [22]
During natural dengue infection in humans themosquito delivers virus in skin epithelium where it infectsand replicates in the cells of mononuclear lineage like mono-cytes dendritic cells macrophages and Langerhans cells[23 24] These infected cells carry the virus to lymph nodeswhere it replicates resulting in viremia which is followedby systemic infection of liver lungs and spleen Howeverin mosquitoes the primary target of DENV infection is theepithelium of the midgut where it first replicates and then
[25 26] spreads to and replicates in salivary glands fromwhere the infection is transmitted through saliva to the nextvertebrate host during the blood meal
3 Dengue Disease
31 Classification The spectrum of clinical illness may rangefrom asymptomatic disease to a broad range of syndromeswith severe clinical manifestations Symptomatic infectionmay range frommild debilitating DF to life threatening DHFand DSS due to plasma leakage in DHF patients Thesethree conditions likely represent progressively severe stagesof a continuous dengue disease spectrum [27] They arebased on traditional WHO classification case definitions andcontinue to be recognized in many regions of the worlddespite the introduction of a new classification system Thenew classification based on a single parameter [5] allowsbetter case capture [28] but is not compatible with restrictedhealth care facilities in endemic regions especially duringoutbreaks [29]
32 DF DF is a self-limiting fever lasting usually for 5ndash7days It is sometimes debilitating during the acute illnessstage The clinical features of DF vary according to the ageof the patient The infants and young children may haveundifferentiated febrile sickness with maculopapular rashThe older children and adults may have mild febrile syn-drome or severe disease with high fever (usually bipha-sic) severe headache retroorbital pain myalgia arthralgianausea vomiting and petechiae Leukopenia and thrombo-cytopenia are usually observed in all ages In some casesDF may accompany bleeding complication such as gingivalbleeding epistaxis gastrointestinal bleeding haematuriaand menorrhagia (in case of women) [27]
33 DHFDSS DHF is characterized by symptoms of DFalong with thrombocytopenia hemorrhagic manifestationsand plasma leakage A positive tourniquet test may be sug-gestive of DHF however this is being debated now due to itslow sensitivityspecificity Plasma leakage determines diseaseseverity in DHF It is also the most important differencebetween DHF and DF Depending on disease severity andclinical manifestations DHF is divided into four grades Ito IV with grade IV being the most severe Several patientsalso have fine petechiae scattered on the extremities axillaeface and soft palate usually seen in the febrile periodThe critical phase is usually reached at the end of febrileillness marked by rapid decrease in temperature and oftenaccompanied by circulatory disturbances including plasmaleakage hemoconcentration and thrombocytopenia [27]
In severe cases with critical plasma loss DSS ensuesand may be life threatening if not treated properly DSS ischaracterized by a rapid weak pulse with narrowing pulsepressure (lt20mmofHg) cold clammy skin and restlessnessThe patient may die within 12ndash24 h of going into shock orrecover rapidly with volume replacement therapy
34 Primary and Secondary Dengue Infection The first expo-sure of an individual to any of the four dengue virus serotypes
Journal of Immunology Research 5
Mature dengue virion
GRP78Mannose receptor
Heparan sulfate
TAMTIM
HSP 7090NKp44DC-SIGN
Clathrin coated pit Internalisation via endocytic pathway
(A) (B)
PM
Outside
Cytoplasm
Figure 4 Schematic representation of the dengue virus entry process The dengue virus makes use of membrane receptors and attachmentfactors on the cell plasma membrane (PM) to find its way to the cytoplasm The mature virion either gets attached directly to a cellularmembrane receptor (a) or uses several attachment factors (b) to finally trigger the endocytic clathrin dependent pathway The endocyticvesicle becomes a late endosome where acidification triggers conformational changes on the E protein dimers to become fusogenic trimersFinally pores are formed and the genome of the virus is released into the cytoplasm
is known as primary dengue infection It maymay not resultin symptomatic infection In primary infection high titersof immunoglobulin M (IgM) and immunoglobulin G (IgG)antibodies appear in 3ndash5 and 6ndash10 days respectively afterthe onset of infection The presence of IgM is transientdisappearing in 2-3 months after the onset of illness whereasIgG persists for life [30] Hence primary infection with aparticular serotype provides life-long immunity against thatserotype But it does not provide continued cross-protectiveimmunity against the remaining serotypes
A secondary infection with a previously unencounteredDENV serotype usually results in classical DF However 2-3of secondary infection cases develop intoDHFwhichmayprogress to DSS and death During a second infection with adifferent serotype the presence of low amounts of heterotypicantibodies (which form complexes with DENVs) promotesthe access of the virus to monocytes via Fc receptors leadingto an increase in viral load and severity of the disease Thisphenomenon is known as ADE The major players of thisphenomenon are cross-reactive antibodies elicited againstthe fusion loop and prM which are found to be weaklyneutralizing leading to enhancement of infection at lowconcentrations [16 21] Although ADE has been found toresult in disease severity all the severe cases are not associatedwith secondary infection nor do all the cases of secondaryinfection progress to DHFDSS [2] In addition to humoralimmunity cross-reactive memory T cells could also playa role in either providing protective immunity or causingimmunopathology [31]
35 Diagnosis and Clinical Management Dengue infectionis usually confirmed by identification of viral genomic RNAantigens or the antibodies it elicits Antigen detection testsbased on NS1 detection have been designed to detect thedengue viral NS1 proteinwhich gets released from the dengueinfected cells and appears early in the bloodstream A 3-in-1test for simultaneous detection of NS1 IgM and IgG is nowavailable ELISA-based serological tests are easy to performand are cost-effective for dengue detection
Up to date there is no antiviral drug available fordengue Treatment is usually based on symptoms and isperformed through medical support For uncomplicatedcases of dengue fever the treatment prescribed is bed restoral rehydration and paracetamol as an antipyretic andanalgesic Patientrsquos health is monitored through variousblood tests from fever day 3 onwards till the conditionimproves Clinical signs that signal progression to seriousdisease include cold limb extremities low pulse low urineoutput signs of mucosal bleeding and abdominal pain DHFis indicated by a rising hematocrit (ge20) and a fallingplatelet count (gt100000mm3) If any of these signs aredetected immediate hospitalization is necessary Treatmentfor DHF patients is based on intravenous fluid therapy tomaintain effective circulation during plasma leakage pluscareful clinical monitoring of hematocrit platelet countpulse rate and blood pressure temperature urine outputfluid administered and other signs of shock Patients usuallyrecover within 12ndash48 h of fluid therapy Treatment for DSSpatients mainly consists of immediate fluid therapy with
Figure 5 Classification of dengue vaccine candidates
colloids and extensive monitoring of any complicationsIn worse case such as internal hemorrhage whole bloodtransfusion may be carried out [27]
4 Dengue Vaccine Strategies
Despite the existing challenges for an ideal dengue vaccinedevelopment of dengue vaccine candidates has progressedover the last decade and some of these have entered clinicaltrials in both endemic and nonendemic areas A classificationof the current approaches for dengue vaccine development isshown in Figure 5
41 Replicating Viral Vaccines These include live-attenuatedviruses (LAV) that are created by reducing the virulence of apathogen without compromising its viability Current meth-ods of producing live-attenuated viruses for dengue vaccinesinclude attenuation by serial passage in cell lines and targetedmutagenesis and by constructing chimeric vaccine viruses
(i) Advantages robust lasting and broad immunity andlower production cost
(ii) Disadvantages difficulty in attenuation genetic insta-bility possibility of reversion and interference in thecase of multicomponent LAV vaccines
411 Cell Culture Passage Based LAV Development of LAVby serial passage in cell lines was started at Mahidol Univer-sity Bangkok Thailand A tetravalent formulation was madeby attenuating all fourDENV serotypes but the vaccine failedto elicit a balanced immune response despite modulating theviral concentrations [32 33] Increased frequency of adversereactions like fever rash myalgia and retroorbital painprimarily related to theDENV-3 vaccine strain was observedFurther development of these LAV strains was stalled [34]
Another LAV based on passaging in cell culture wasdeveloped by Walter Reed Army Institute of Research(WRAIR) Maryland USA and is being evaluated in clinical
trials in collaboration with GlaxoSmithKline (GSK) Allfour DENV serotypes were attenuated by passaging in pri-mary dog kidney (PDK) cells and a tetravalent formulation(F17Pre) was developed which was found to result in DENV-4 vaccine-induced viremia during phase II clinical trials[35 36] In a separate phase II randomized observer-blindplacebo-controlled trial in 86 healthy flavivirus naıve adultsin USA F17Pre DENVs were rederived and passaged infetus rhesus lung cells to obtain seed viruses of higher purityResultant formulations F17 and F19 containing equivalentamounts of vaccine components except DENV-4 being 10-fold higher in F17 were evaluated An acceptable safetyand immunogenicity profile was observed after 2 doses ofLAV with tetravalent antibody rates of 60 and 67 inparticipants receiving F17 and F19 respectively It was alsoreported that although F19 was formulated to contain 10-foldless DENV-4 it was found to be only fourfold less at the timeof vaccine release The neutralization titers against DENV-4 were found to be comparable at 70 and 46 for F17 andF19 respectively Notably the incidence of DENV-4 vaccine-induced viremia reduced (with only one case in F17 group)probably due to rederivation and passage which attenuatedthe DENV-4 strain further [37] In a similar phase II trialin healthy children and adults in Puerto Rico F17 and F19were evaluated againTheDENV-4 in vitropotency in F19wasfound to be 50-fold less instead of being 10-fold according toformulation design Thus there have been issues related tostorage stability of DENV-4 strain [38]
412 Targeted Mutagenesis Based Live-Attenuated VaccineThis strategy was first successfully explored by the Laboratoryof Infectious Disease at the National Institute of Allergy andInfectious Disease (NIAID) National Institutes of Health(NIH) Maryland USA NIH has established nonexclusivelicense with manufacturers in Brazil (Instituto Butantan)Vietnam (Vabiotech) and India (Serum Institute of Indiaand Panacea Biotech) for its development This vaccinecandidate is amixture of fourDENV strains attenuated by site
Journal of Immunology Research 7
directed mutagenesis to delete 30 nucleotides in the 31015840UTRDENV-1 and DENV-4 attenuated strains were designatedas DEN1Δ30 and DEN4Δ30 respectively [39] DENV-2 andDENV-3 attenuated strains were made by using DEN4Δ30as a backbone and replacing their structural prM and Egenes with those of the corresponding serotype Notablychimerization resulted in overattenuation of rDEN24Δ30and rDEN34Δ30 strainsTheDENV-3 component wasmod-ified variably and rDEN3Δ3031 strain was selected whereadditional 31 nucleotides were deleted from rDEN3Δ30Infectivity of DENV-2 component has been improved intetravalent formulation TV005 by using DENV-2 attenuatedrDEN24Δ30 strain at a 10-fold higher dose (104 pfu) thanother components (103 pfu) tetravalent formulation TV003contains 103 pfu of each of the four components Importantlya single dose of TV005 has been found to be efficacious inproviding sterilizing immunity Additionally TV003TV005are being evaluated in a human challenge model to enable amore stringent assessment of its protective efficacy TV003has been found to protect vaccinees against challenge withDENV-2 attenuated rDEN2Δ30 strain [40 41] Similar evalu-ation of protective efficacy is ongoing for TV005 andDENV-3human challenge experiments are being planned [40] PhaseIII of this vaccine candidate has begun in Brazil [42]
413 Chimeric Dengue Vaccine Chimeric dengue vaccineshave been designed using two approaches (i) with anotherattenuated flavivirus and (ii) with an attenuated DENV strain(intertypic chimera) The vaccine where chimera of DENVhas been made with another flavivirus is the chimeric yellowfever-dengue (CYD) vaccine which is being developed bySanofi Pasteur and licensed under the brand name ldquoDeng-vaxiardquo [4] In this vaccine prM and E genes of the attenuatedyellow fever LAV strain 17D have been replaced with thecorresponding genes from DENV [43] The rationale behindthis design was the fact that humoral response against thestructural proteins of dengue was responsible for protectiveimmunity during natural infection and thus these chimeraswould generate a protective immune response in vaccineesA tetravalent mixture of the four chimeric viruses hasundergone extensive clinical evaluation and has recently beenapproved in Mexico Brazil El Salvador and Philippines[4 44] This vaccine will be discussed in detail in the latersections
An example of intertypic chimera is DENVax developedby Inviragen Inc Fort Collins CO USA DENV-2 strainattenuated by 53 passages in PDK cells (made at MahidolUniversity) has been used as a backbone for generatingchimera prMandE gene of this strainwere replaced by corre-sponding genes fromDENV-1DENV-3 andDENV-4As themutations in the attenuated strain were in the nonstructuralproteins this strain was used as such for DENV-2 componentin the tetravalent formulationThese chimeric viruses showeda temperature-sensitive phenotype reduced replication inmosquito cell lines high degree of genetic stability and lackof neurovirulence in sucklingmice [45]Three tetravalent for-mulations with variable dose of each component were evalu-ated in nonhuman primates It was observed that DENV-2
was the dominating component and its replicative potentialreduced by increasing the DENV-3 andDENV-4 componentThis variation in DENV-2 induced viremia due to the varia-tion in the dose of DENV-3 and DENV-4 components indi-cated viral interference Moreover the neutralizing antibodytiters were found to be significantly low against DENV-4 anddespite this macaques were found to be protected againstDENV-4 challenge [46] A phase I clinical trial of low andhigh doses of the DENVax in healthy subjects in Columbiarevealed that the candidate was safe and immunogenicNotably it corroborated the findings made in nonhuman pri-mate study that neutralizing antibody titers elicited by DEN-Vax are lowest against DENV-4 and highest against DENV-2[47] Further insights into its efficacy will be revealed throughits phase II clinical trial evaluation Meanwhile phase IIIevaluation of this vaccine has now been initiated [48]
42 Nonreplicating Viral Vaccines These vaccine candidatesare not capable of replicating and thus offer the advantage ofconferring immunity without the risk of infection There aremultiple strategies to develop this class of vaccines like DNAvaccines subunit proteins VLPs and so forth
(i) Advantages reduced reactogenicity better suitabilityfor immune-compromised individuals and balancedimmune response in case of tetravalent formulation
(ii) Disadvantages less broad potent and durable im-mune response whichmay result in ADE and requir-ing the use of adjuvants
421 Purified Inactivated Virus (PIV) WRAIR MarylandUSA developed an inactivated monovalent dengue vaccineby formalin treatment It was found to be safe and immuno-genic in mice and Rhesus macaques [49 50] Althoughsuch vaccines would not show viral interference or revertto a pathogenic strain their use as the sole immunizationapproach is limited because of conformational changes invirus by formalin treatment and lack of replication Howeverthis vaccine has been tested as the priming vaccine in a prime-boost immunization strategy with a LAV as the boostervaccine leading to complete protection in macaques [51]Phase I trial evaluating the safety of 25 and 5 120583g of DENV-1component administered on days 0 and 28 in flavivirus naıvepopulation in the USA has been completed [52] Two phase Itrials evaluating tetravalent mixture of the four PIVs (TPIV1 120583g of each of the four PIVs) are being evaluated with alumand two proprietory adjuvants of GSK (AS01E1 and AS03B1)in healthy adults in the USA [53] and in Puerto Rico [54]Healthy adults in the USA are also being recruited in anotherphase I study where TPIValum is being evaluated in prime-boot vaccination with WRAIRGSKrsquos tetravalent LAV [55]
422 Recombinant Subunit Vaccine Recombinant E proteinsof dengue have been expressed in yeast and insect expres-sion systems and have been analyzed for vaccine efficacyin mice and monkeys All these studies have focused onthe DENV E aminoterminal 80 of the molecule knownas the ectodomain Deletion of 20 of the E protein at
8 Journal of Immunology Research
the C-terminal which is a transmembrane region allowsextracellular secretion and easy purification while retainingits antigenicityThe recombinant 80 E proteins also knownas r80E of the four DENV serotypes are being manufac-tured by Hawaii Biotech Inc HI USA and Merck andCo NJ USA Monovalent DEN2 80E was evaluated witha panel of adjuvants in mice and saponin-based adjuvantISCOMATRIXtrade was found to be the most immunogenicimmunogenicity with alum as adjuvant was poor Thiswas followed by evaluation of tetravalent formulation withISCOMATRIX in macaques where titers against DENV-4were found to be the weakest [56] To overcome the lowimmunogenicity of DEN4 80E its dimeric form and doubledose were explored in macaques which led to comparableimprovement in the neutralizing titers against DENV-4Though the titers against DENV-4 improved they were lowerthan the titers against DENV-1 DENV-2 and DENV-3 [57]Based on these results a tetravalent mixture of the four r80Escontaining 10 10 10 and 20120583g of DEN1 DEN2 DEN3and DEN4 80E respectively was further evaluated in flavi-naıve and dengue-primed macaques where it was found togenerate a more balanced immune response against the fourserotypes in 0- 1- and 6-month immunization schedule ascompared to 0 1 and 2 months Moreover two doses (10and 50 120583g) of DEN1 80Ealum administered in flavi-naıveadults on 0 1 and 2 months were found to be safe Howeverit elicited only modest DENV-1 neutralizing titers whichwaned almost completely 26 weeks after the final dose [58]A phase I study examining safety and immunogenicity oftetravalent formulation with and without adjuvant (alum andISCOMATRIX) in healthy adults has been completed [59]
Recombinant antigens based on DENV EDIII have beenproduced by different groups using E coli and yeast expres-sion hosts Recombinant EDIII antigens expressed eitherindependently or fused to different carriers such as maltose-binding protein [60] and the Neisseria meningitides p64kprotein have been shown to generate anti-DENV immuneresponses in mice and nonhuman primates [61ndash63] Thesevaccines candidates are in preclinical phase currently
423 Dengue DNA Vaccine This vaccine consists of a plas-mid vector containing the gene(s) encoding for an antigenwhich on immunization is taken up by antigen presentingcells (APCs) Once the plasmid enters the cell it codes forthe antigen which finally gets associated with MHC classI molecules and gets displayed on the cell surface induc-ing protective cytotoxic immune response Naval MedicalResearch Center (NMRC) USA has developed a DENV-1DNA vaccine candidate (D1ME100) by cloning prM and Egene of DENV-1 serotype into plasmid vector which wasextensively evaluated inmice andmacaques without adjuvant[64] before phase 1 trials in healthy adults AlthoughDENV-1DNA vaccine was found to be well tolerated the neutralizingantibody titers and the number of responders were foundto be low [64 65] Thus to enhance its immunogenicitya lipid-based adjuvant Vaxfectin was explored Tetravalentdengue DNA vaccine (TVDV) was evaluated for immuno-genicity with and without Vaxfectin in macaques It wasobserved that Vaxfectin resulted in higher and more stable
(evaluated till 6 months after final boost) titers The averageneutralization titers with TVDVVaxfectin against DENV-1DENV-2 DENV-3 and DENV-4 a month after final boostwere approximately 200 270 170 and 70 respectively Sixmonths after the final boost the titers against DENV-2 andDENV-3 reduced while those against DENV-1 and DENV-4increased marginally In the group without Vaxfectin titersagainst DENV-2 only were detectable 6 months after the finalboost Moreover Vaxfectin allowed better protection fromviremia against DENV-2 challenge [66] After establishingnontoxicity of TVDVVaxfectin in New Zealand white rab-bits [67] a phase I trial was initiated in 2011 in USA [68]
424 Replication-Defective Virus Vectored Vaccines In thisapproach a virus is used as a vector to carry antigenic genesthat are capable of eliciting neutralizing antibody responseSome examples of viral vectors are adenovirus vectors Ven-ezuelan equine encephalitis virus vector and attenuatedmeasles virus [69ndash71] An example of virus vectored denguevaccine is cAdVax It consists of bivalent constructs express-ing prM and E proteins from two dengue serotypes each(DENV-1 and DENV-3 together in one and DENV-2 andDENV-4 in another construct) Study in NHPs showedproduction of neutralizing antibodies to respective DENVserotypes [72 73] Therefore a tetravalent formulation (cAd-Vax-DenTV) was prepared bymixing the bivalent constructswhich showed protection against all serotypes on DENVchallenge in Rhesus macaques [69]
425 Virus Like Particle (VLP) Vaccines The prM and Eproteins of DENVs coexpressed in heterologous hosts havebeen shown to coassemble into VLPs Thus a vaccine basedon physical mixtures of four monovalent DENV VLPs canbe developed to have a tetravalent formulation [74] Fromthe perspective of using VLPs for vaccine purpose the yeastsystemmay bemore suitable as it has the potential for higheryields and can glycosylate the antigens Recent work indicatesthat yeast expressed-DENV E ectodomain forms VLPs in theabsence of prM [75] Another approach is based on displayingthe DENV EDIII on VLPs formed by hepatitis B virus coreantigens [27]
5 Dengvaxia
Dengue vaccine candidates which have reached clinical trialsare given in Table 2 It is worthwhile to discuss the front-runner CYD vaccine developed by Sanofi Pasteur whichhas recently been approved as Dengvaxia in Mexico BrazilEl Salvador and Philippines [4] Dengvaxia is a tetravalentdengue chimeric live-attenuated virus vaccine based onlicensed yellow fever vaccine 17D It was constructed byreplacement of structural genes of live-attenuated yellowfever virus vaccine 17D with structural genes from eachDENV serotype [76]
51 Preclinical Data The immunogenicity of various tetrava-lent formulations of the chimeric viruses was evaluated inmacaques which revealed immunodominance of serotype
Journal of Immunology Research 9
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
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[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
Figure 3 DENV-1 DENV-2 DENV-3 and DENV-4 with EDIII(circled) in red green blue and black respectively EDIIIs elicitstrongly neutralizing serotype-specific antibodies (antibodies inbold) Owing to homology especially in domains (EDIII and prM)in yellow cross-reactive weakly neutralizing antibodies (unboldantibodies) and nonneutralizing enhancing antibodies (dashedantibodies) are elicited in bulk
It has been reported that it is serotype-specific neutralizingantibodies and not cross-reactive neutralizing antibodiesthat confer protection against infection [20] It has beenreported that bulk of the immune response is elicitedagainst the cross-reactive domains of EDI EDII and prM(yellow domain in the virus image) [16 21] Anti-EDIIIantibodies (Figure 3 unbold antibodies) are largely het-erotypic weaklynonneutralizing while prM antibodies (Fig-ure 3 dashed antibodies) are largely nonneutralizing andcross-reactive It is believed that the virus utilizes suchweaknonneutralizing cross-reactive antibodies in gainingaccess into the host cell via Fc receptor as an alternativepathway during a secondary infection with a heterologousserotype leading to enhancement of infection [16 20 21]This phenomenon is known as antibody-dependent enhance-ment (ADE)
Thus it can be inferred that strongly neutralizing anti-bodies prevent ADE caused by a larger population of weaklynonneutralizing enhancing antibodies A vaccine candidatecapable of a larger population of strongly neutralizing anti-bodies could probably be an ideal vaccine showcasing strongprotection without ADE This can probably be achieved bydesigning the vaccine candidate and not by default strategy
23 Cellular Targets and Receptor Interaction The currentmodel of flavivirus cell entry suggests the use of two func-tionally different sets of molecules attachment factors thathelp the virus to concentrate on the cell surface and primaryreceptor(s) which help directing the virion to the endocyticpathway (Figure 4) [22]
During natural dengue infection in humans themosquito delivers virus in skin epithelium where it infectsand replicates in the cells of mononuclear lineage like mono-cytes dendritic cells macrophages and Langerhans cells[23 24] These infected cells carry the virus to lymph nodeswhere it replicates resulting in viremia which is followedby systemic infection of liver lungs and spleen Howeverin mosquitoes the primary target of DENV infection is theepithelium of the midgut where it first replicates and then
[25 26] spreads to and replicates in salivary glands fromwhere the infection is transmitted through saliva to the nextvertebrate host during the blood meal
3 Dengue Disease
31 Classification The spectrum of clinical illness may rangefrom asymptomatic disease to a broad range of syndromeswith severe clinical manifestations Symptomatic infectionmay range frommild debilitating DF to life threatening DHFand DSS due to plasma leakage in DHF patients Thesethree conditions likely represent progressively severe stagesof a continuous dengue disease spectrum [27] They arebased on traditional WHO classification case definitions andcontinue to be recognized in many regions of the worlddespite the introduction of a new classification system Thenew classification based on a single parameter [5] allowsbetter case capture [28] but is not compatible with restrictedhealth care facilities in endemic regions especially duringoutbreaks [29]
32 DF DF is a self-limiting fever lasting usually for 5ndash7days It is sometimes debilitating during the acute illnessstage The clinical features of DF vary according to the ageof the patient The infants and young children may haveundifferentiated febrile sickness with maculopapular rashThe older children and adults may have mild febrile syn-drome or severe disease with high fever (usually bipha-sic) severe headache retroorbital pain myalgia arthralgianausea vomiting and petechiae Leukopenia and thrombo-cytopenia are usually observed in all ages In some casesDF may accompany bleeding complication such as gingivalbleeding epistaxis gastrointestinal bleeding haematuriaand menorrhagia (in case of women) [27]
33 DHFDSS DHF is characterized by symptoms of DFalong with thrombocytopenia hemorrhagic manifestationsand plasma leakage A positive tourniquet test may be sug-gestive of DHF however this is being debated now due to itslow sensitivityspecificity Plasma leakage determines diseaseseverity in DHF It is also the most important differencebetween DHF and DF Depending on disease severity andclinical manifestations DHF is divided into four grades Ito IV with grade IV being the most severe Several patientsalso have fine petechiae scattered on the extremities axillaeface and soft palate usually seen in the febrile periodThe critical phase is usually reached at the end of febrileillness marked by rapid decrease in temperature and oftenaccompanied by circulatory disturbances including plasmaleakage hemoconcentration and thrombocytopenia [27]
In severe cases with critical plasma loss DSS ensuesand may be life threatening if not treated properly DSS ischaracterized by a rapid weak pulse with narrowing pulsepressure (lt20mmofHg) cold clammy skin and restlessnessThe patient may die within 12ndash24 h of going into shock orrecover rapidly with volume replacement therapy
34 Primary and Secondary Dengue Infection The first expo-sure of an individual to any of the four dengue virus serotypes
Journal of Immunology Research 5
Mature dengue virion
GRP78Mannose receptor
Heparan sulfate
TAMTIM
HSP 7090NKp44DC-SIGN
Clathrin coated pit Internalisation via endocytic pathway
(A) (B)
PM
Outside
Cytoplasm
Figure 4 Schematic representation of the dengue virus entry process The dengue virus makes use of membrane receptors and attachmentfactors on the cell plasma membrane (PM) to find its way to the cytoplasm The mature virion either gets attached directly to a cellularmembrane receptor (a) or uses several attachment factors (b) to finally trigger the endocytic clathrin dependent pathway The endocyticvesicle becomes a late endosome where acidification triggers conformational changes on the E protein dimers to become fusogenic trimersFinally pores are formed and the genome of the virus is released into the cytoplasm
is known as primary dengue infection It maymay not resultin symptomatic infection In primary infection high titersof immunoglobulin M (IgM) and immunoglobulin G (IgG)antibodies appear in 3ndash5 and 6ndash10 days respectively afterthe onset of infection The presence of IgM is transientdisappearing in 2-3 months after the onset of illness whereasIgG persists for life [30] Hence primary infection with aparticular serotype provides life-long immunity against thatserotype But it does not provide continued cross-protectiveimmunity against the remaining serotypes
A secondary infection with a previously unencounteredDENV serotype usually results in classical DF However 2-3of secondary infection cases develop intoDHFwhichmayprogress to DSS and death During a second infection with adifferent serotype the presence of low amounts of heterotypicantibodies (which form complexes with DENVs) promotesthe access of the virus to monocytes via Fc receptors leadingto an increase in viral load and severity of the disease Thisphenomenon is known as ADE The major players of thisphenomenon are cross-reactive antibodies elicited againstthe fusion loop and prM which are found to be weaklyneutralizing leading to enhancement of infection at lowconcentrations [16 21] Although ADE has been found toresult in disease severity all the severe cases are not associatedwith secondary infection nor do all the cases of secondaryinfection progress to DHFDSS [2] In addition to humoralimmunity cross-reactive memory T cells could also playa role in either providing protective immunity or causingimmunopathology [31]
35 Diagnosis and Clinical Management Dengue infectionis usually confirmed by identification of viral genomic RNAantigens or the antibodies it elicits Antigen detection testsbased on NS1 detection have been designed to detect thedengue viral NS1 proteinwhich gets released from the dengueinfected cells and appears early in the bloodstream A 3-in-1test for simultaneous detection of NS1 IgM and IgG is nowavailable ELISA-based serological tests are easy to performand are cost-effective for dengue detection
Up to date there is no antiviral drug available fordengue Treatment is usually based on symptoms and isperformed through medical support For uncomplicatedcases of dengue fever the treatment prescribed is bed restoral rehydration and paracetamol as an antipyretic andanalgesic Patientrsquos health is monitored through variousblood tests from fever day 3 onwards till the conditionimproves Clinical signs that signal progression to seriousdisease include cold limb extremities low pulse low urineoutput signs of mucosal bleeding and abdominal pain DHFis indicated by a rising hematocrit (ge20) and a fallingplatelet count (gt100000mm3) If any of these signs aredetected immediate hospitalization is necessary Treatmentfor DHF patients is based on intravenous fluid therapy tomaintain effective circulation during plasma leakage pluscareful clinical monitoring of hematocrit platelet countpulse rate and blood pressure temperature urine outputfluid administered and other signs of shock Patients usuallyrecover within 12ndash48 h of fluid therapy Treatment for DSSpatients mainly consists of immediate fluid therapy with
Figure 5 Classification of dengue vaccine candidates
colloids and extensive monitoring of any complicationsIn worse case such as internal hemorrhage whole bloodtransfusion may be carried out [27]
4 Dengue Vaccine Strategies
Despite the existing challenges for an ideal dengue vaccinedevelopment of dengue vaccine candidates has progressedover the last decade and some of these have entered clinicaltrials in both endemic and nonendemic areas A classificationof the current approaches for dengue vaccine development isshown in Figure 5
41 Replicating Viral Vaccines These include live-attenuatedviruses (LAV) that are created by reducing the virulence of apathogen without compromising its viability Current meth-ods of producing live-attenuated viruses for dengue vaccinesinclude attenuation by serial passage in cell lines and targetedmutagenesis and by constructing chimeric vaccine viruses
(i) Advantages robust lasting and broad immunity andlower production cost
(ii) Disadvantages difficulty in attenuation genetic insta-bility possibility of reversion and interference in thecase of multicomponent LAV vaccines
411 Cell Culture Passage Based LAV Development of LAVby serial passage in cell lines was started at Mahidol Univer-sity Bangkok Thailand A tetravalent formulation was madeby attenuating all fourDENV serotypes but the vaccine failedto elicit a balanced immune response despite modulating theviral concentrations [32 33] Increased frequency of adversereactions like fever rash myalgia and retroorbital painprimarily related to theDENV-3 vaccine strain was observedFurther development of these LAV strains was stalled [34]
Another LAV based on passaging in cell culture wasdeveloped by Walter Reed Army Institute of Research(WRAIR) Maryland USA and is being evaluated in clinical
trials in collaboration with GlaxoSmithKline (GSK) Allfour DENV serotypes were attenuated by passaging in pri-mary dog kidney (PDK) cells and a tetravalent formulation(F17Pre) was developed which was found to result in DENV-4 vaccine-induced viremia during phase II clinical trials[35 36] In a separate phase II randomized observer-blindplacebo-controlled trial in 86 healthy flavivirus naıve adultsin USA F17Pre DENVs were rederived and passaged infetus rhesus lung cells to obtain seed viruses of higher purityResultant formulations F17 and F19 containing equivalentamounts of vaccine components except DENV-4 being 10-fold higher in F17 were evaluated An acceptable safetyand immunogenicity profile was observed after 2 doses ofLAV with tetravalent antibody rates of 60 and 67 inparticipants receiving F17 and F19 respectively It was alsoreported that although F19 was formulated to contain 10-foldless DENV-4 it was found to be only fourfold less at the timeof vaccine release The neutralization titers against DENV-4 were found to be comparable at 70 and 46 for F17 andF19 respectively Notably the incidence of DENV-4 vaccine-induced viremia reduced (with only one case in F17 group)probably due to rederivation and passage which attenuatedthe DENV-4 strain further [37] In a similar phase II trialin healthy children and adults in Puerto Rico F17 and F19were evaluated againTheDENV-4 in vitropotency in F19wasfound to be 50-fold less instead of being 10-fold according toformulation design Thus there have been issues related tostorage stability of DENV-4 strain [38]
412 Targeted Mutagenesis Based Live-Attenuated VaccineThis strategy was first successfully explored by the Laboratoryof Infectious Disease at the National Institute of Allergy andInfectious Disease (NIAID) National Institutes of Health(NIH) Maryland USA NIH has established nonexclusivelicense with manufacturers in Brazil (Instituto Butantan)Vietnam (Vabiotech) and India (Serum Institute of Indiaand Panacea Biotech) for its development This vaccinecandidate is amixture of fourDENV strains attenuated by site
Journal of Immunology Research 7
directed mutagenesis to delete 30 nucleotides in the 31015840UTRDENV-1 and DENV-4 attenuated strains were designatedas DEN1Δ30 and DEN4Δ30 respectively [39] DENV-2 andDENV-3 attenuated strains were made by using DEN4Δ30as a backbone and replacing their structural prM and Egenes with those of the corresponding serotype Notablychimerization resulted in overattenuation of rDEN24Δ30and rDEN34Δ30 strainsTheDENV-3 component wasmod-ified variably and rDEN3Δ3031 strain was selected whereadditional 31 nucleotides were deleted from rDEN3Δ30Infectivity of DENV-2 component has been improved intetravalent formulation TV005 by using DENV-2 attenuatedrDEN24Δ30 strain at a 10-fold higher dose (104 pfu) thanother components (103 pfu) tetravalent formulation TV003contains 103 pfu of each of the four components Importantlya single dose of TV005 has been found to be efficacious inproviding sterilizing immunity Additionally TV003TV005are being evaluated in a human challenge model to enable amore stringent assessment of its protective efficacy TV003has been found to protect vaccinees against challenge withDENV-2 attenuated rDEN2Δ30 strain [40 41] Similar evalu-ation of protective efficacy is ongoing for TV005 andDENV-3human challenge experiments are being planned [40] PhaseIII of this vaccine candidate has begun in Brazil [42]
413 Chimeric Dengue Vaccine Chimeric dengue vaccineshave been designed using two approaches (i) with anotherattenuated flavivirus and (ii) with an attenuated DENV strain(intertypic chimera) The vaccine where chimera of DENVhas been made with another flavivirus is the chimeric yellowfever-dengue (CYD) vaccine which is being developed bySanofi Pasteur and licensed under the brand name ldquoDeng-vaxiardquo [4] In this vaccine prM and E genes of the attenuatedyellow fever LAV strain 17D have been replaced with thecorresponding genes from DENV [43] The rationale behindthis design was the fact that humoral response against thestructural proteins of dengue was responsible for protectiveimmunity during natural infection and thus these chimeraswould generate a protective immune response in vaccineesA tetravalent mixture of the four chimeric viruses hasundergone extensive clinical evaluation and has recently beenapproved in Mexico Brazil El Salvador and Philippines[4 44] This vaccine will be discussed in detail in the latersections
An example of intertypic chimera is DENVax developedby Inviragen Inc Fort Collins CO USA DENV-2 strainattenuated by 53 passages in PDK cells (made at MahidolUniversity) has been used as a backbone for generatingchimera prMandE gene of this strainwere replaced by corre-sponding genes fromDENV-1DENV-3 andDENV-4As themutations in the attenuated strain were in the nonstructuralproteins this strain was used as such for DENV-2 componentin the tetravalent formulationThese chimeric viruses showeda temperature-sensitive phenotype reduced replication inmosquito cell lines high degree of genetic stability and lackof neurovirulence in sucklingmice [45]Three tetravalent for-mulations with variable dose of each component were evalu-ated in nonhuman primates It was observed that DENV-2
was the dominating component and its replicative potentialreduced by increasing the DENV-3 andDENV-4 componentThis variation in DENV-2 induced viremia due to the varia-tion in the dose of DENV-3 and DENV-4 components indi-cated viral interference Moreover the neutralizing antibodytiters were found to be significantly low against DENV-4 anddespite this macaques were found to be protected againstDENV-4 challenge [46] A phase I clinical trial of low andhigh doses of the DENVax in healthy subjects in Columbiarevealed that the candidate was safe and immunogenicNotably it corroborated the findings made in nonhuman pri-mate study that neutralizing antibody titers elicited by DEN-Vax are lowest against DENV-4 and highest against DENV-2[47] Further insights into its efficacy will be revealed throughits phase II clinical trial evaluation Meanwhile phase IIIevaluation of this vaccine has now been initiated [48]
42 Nonreplicating Viral Vaccines These vaccine candidatesare not capable of replicating and thus offer the advantage ofconferring immunity without the risk of infection There aremultiple strategies to develop this class of vaccines like DNAvaccines subunit proteins VLPs and so forth
(i) Advantages reduced reactogenicity better suitabilityfor immune-compromised individuals and balancedimmune response in case of tetravalent formulation
(ii) Disadvantages less broad potent and durable im-mune response whichmay result in ADE and requir-ing the use of adjuvants
421 Purified Inactivated Virus (PIV) WRAIR MarylandUSA developed an inactivated monovalent dengue vaccineby formalin treatment It was found to be safe and immuno-genic in mice and Rhesus macaques [49 50] Althoughsuch vaccines would not show viral interference or revertto a pathogenic strain their use as the sole immunizationapproach is limited because of conformational changes invirus by formalin treatment and lack of replication Howeverthis vaccine has been tested as the priming vaccine in a prime-boost immunization strategy with a LAV as the boostervaccine leading to complete protection in macaques [51]Phase I trial evaluating the safety of 25 and 5 120583g of DENV-1component administered on days 0 and 28 in flavivirus naıvepopulation in the USA has been completed [52] Two phase Itrials evaluating tetravalent mixture of the four PIVs (TPIV1 120583g of each of the four PIVs) are being evaluated with alumand two proprietory adjuvants of GSK (AS01E1 and AS03B1)in healthy adults in the USA [53] and in Puerto Rico [54]Healthy adults in the USA are also being recruited in anotherphase I study where TPIValum is being evaluated in prime-boot vaccination with WRAIRGSKrsquos tetravalent LAV [55]
422 Recombinant Subunit Vaccine Recombinant E proteinsof dengue have been expressed in yeast and insect expres-sion systems and have been analyzed for vaccine efficacyin mice and monkeys All these studies have focused onthe DENV E aminoterminal 80 of the molecule knownas the ectodomain Deletion of 20 of the E protein at
8 Journal of Immunology Research
the C-terminal which is a transmembrane region allowsextracellular secretion and easy purification while retainingits antigenicityThe recombinant 80 E proteins also knownas r80E of the four DENV serotypes are being manufac-tured by Hawaii Biotech Inc HI USA and Merck andCo NJ USA Monovalent DEN2 80E was evaluated witha panel of adjuvants in mice and saponin-based adjuvantISCOMATRIXtrade was found to be the most immunogenicimmunogenicity with alum as adjuvant was poor Thiswas followed by evaluation of tetravalent formulation withISCOMATRIX in macaques where titers against DENV-4were found to be the weakest [56] To overcome the lowimmunogenicity of DEN4 80E its dimeric form and doubledose were explored in macaques which led to comparableimprovement in the neutralizing titers against DENV-4Though the titers against DENV-4 improved they were lowerthan the titers against DENV-1 DENV-2 and DENV-3 [57]Based on these results a tetravalent mixture of the four r80Escontaining 10 10 10 and 20120583g of DEN1 DEN2 DEN3and DEN4 80E respectively was further evaluated in flavi-naıve and dengue-primed macaques where it was found togenerate a more balanced immune response against the fourserotypes in 0- 1- and 6-month immunization schedule ascompared to 0 1 and 2 months Moreover two doses (10and 50 120583g) of DEN1 80Ealum administered in flavi-naıveadults on 0 1 and 2 months were found to be safe Howeverit elicited only modest DENV-1 neutralizing titers whichwaned almost completely 26 weeks after the final dose [58]A phase I study examining safety and immunogenicity oftetravalent formulation with and without adjuvant (alum andISCOMATRIX) in healthy adults has been completed [59]
Recombinant antigens based on DENV EDIII have beenproduced by different groups using E coli and yeast expres-sion hosts Recombinant EDIII antigens expressed eitherindependently or fused to different carriers such as maltose-binding protein [60] and the Neisseria meningitides p64kprotein have been shown to generate anti-DENV immuneresponses in mice and nonhuman primates [61ndash63] Thesevaccines candidates are in preclinical phase currently
423 Dengue DNA Vaccine This vaccine consists of a plas-mid vector containing the gene(s) encoding for an antigenwhich on immunization is taken up by antigen presentingcells (APCs) Once the plasmid enters the cell it codes forthe antigen which finally gets associated with MHC classI molecules and gets displayed on the cell surface induc-ing protective cytotoxic immune response Naval MedicalResearch Center (NMRC) USA has developed a DENV-1DNA vaccine candidate (D1ME100) by cloning prM and Egene of DENV-1 serotype into plasmid vector which wasextensively evaluated inmice andmacaques without adjuvant[64] before phase 1 trials in healthy adults AlthoughDENV-1DNA vaccine was found to be well tolerated the neutralizingantibody titers and the number of responders were foundto be low [64 65] Thus to enhance its immunogenicitya lipid-based adjuvant Vaxfectin was explored Tetravalentdengue DNA vaccine (TVDV) was evaluated for immuno-genicity with and without Vaxfectin in macaques It wasobserved that Vaxfectin resulted in higher and more stable
(evaluated till 6 months after final boost) titers The averageneutralization titers with TVDVVaxfectin against DENV-1DENV-2 DENV-3 and DENV-4 a month after final boostwere approximately 200 270 170 and 70 respectively Sixmonths after the final boost the titers against DENV-2 andDENV-3 reduced while those against DENV-1 and DENV-4increased marginally In the group without Vaxfectin titersagainst DENV-2 only were detectable 6 months after the finalboost Moreover Vaxfectin allowed better protection fromviremia against DENV-2 challenge [66] After establishingnontoxicity of TVDVVaxfectin in New Zealand white rab-bits [67] a phase I trial was initiated in 2011 in USA [68]
424 Replication-Defective Virus Vectored Vaccines In thisapproach a virus is used as a vector to carry antigenic genesthat are capable of eliciting neutralizing antibody responseSome examples of viral vectors are adenovirus vectors Ven-ezuelan equine encephalitis virus vector and attenuatedmeasles virus [69ndash71] An example of virus vectored denguevaccine is cAdVax It consists of bivalent constructs express-ing prM and E proteins from two dengue serotypes each(DENV-1 and DENV-3 together in one and DENV-2 andDENV-4 in another construct) Study in NHPs showedproduction of neutralizing antibodies to respective DENVserotypes [72 73] Therefore a tetravalent formulation (cAd-Vax-DenTV) was prepared bymixing the bivalent constructswhich showed protection against all serotypes on DENVchallenge in Rhesus macaques [69]
425 Virus Like Particle (VLP) Vaccines The prM and Eproteins of DENVs coexpressed in heterologous hosts havebeen shown to coassemble into VLPs Thus a vaccine basedon physical mixtures of four monovalent DENV VLPs canbe developed to have a tetravalent formulation [74] Fromthe perspective of using VLPs for vaccine purpose the yeastsystemmay bemore suitable as it has the potential for higheryields and can glycosylate the antigens Recent work indicatesthat yeast expressed-DENV E ectodomain forms VLPs in theabsence of prM [75] Another approach is based on displayingthe DENV EDIII on VLPs formed by hepatitis B virus coreantigens [27]
5 Dengvaxia
Dengue vaccine candidates which have reached clinical trialsare given in Table 2 It is worthwhile to discuss the front-runner CYD vaccine developed by Sanofi Pasteur whichhas recently been approved as Dengvaxia in Mexico BrazilEl Salvador and Philippines [4] Dengvaxia is a tetravalentdengue chimeric live-attenuated virus vaccine based onlicensed yellow fever vaccine 17D It was constructed byreplacement of structural genes of live-attenuated yellowfever virus vaccine 17D with structural genes from eachDENV serotype [76]
51 Preclinical Data The immunogenicity of various tetrava-lent formulations of the chimeric viruses was evaluated inmacaques which revealed immunodominance of serotype
Journal of Immunology Research 9
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
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[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
Clathrin coated pit Internalisation via endocytic pathway
(A) (B)
PM
Outside
Cytoplasm
Figure 4 Schematic representation of the dengue virus entry process The dengue virus makes use of membrane receptors and attachmentfactors on the cell plasma membrane (PM) to find its way to the cytoplasm The mature virion either gets attached directly to a cellularmembrane receptor (a) or uses several attachment factors (b) to finally trigger the endocytic clathrin dependent pathway The endocyticvesicle becomes a late endosome where acidification triggers conformational changes on the E protein dimers to become fusogenic trimersFinally pores are formed and the genome of the virus is released into the cytoplasm
is known as primary dengue infection It maymay not resultin symptomatic infection In primary infection high titersof immunoglobulin M (IgM) and immunoglobulin G (IgG)antibodies appear in 3ndash5 and 6ndash10 days respectively afterthe onset of infection The presence of IgM is transientdisappearing in 2-3 months after the onset of illness whereasIgG persists for life [30] Hence primary infection with aparticular serotype provides life-long immunity against thatserotype But it does not provide continued cross-protectiveimmunity against the remaining serotypes
A secondary infection with a previously unencounteredDENV serotype usually results in classical DF However 2-3of secondary infection cases develop intoDHFwhichmayprogress to DSS and death During a second infection with adifferent serotype the presence of low amounts of heterotypicantibodies (which form complexes with DENVs) promotesthe access of the virus to monocytes via Fc receptors leadingto an increase in viral load and severity of the disease Thisphenomenon is known as ADE The major players of thisphenomenon are cross-reactive antibodies elicited againstthe fusion loop and prM which are found to be weaklyneutralizing leading to enhancement of infection at lowconcentrations [16 21] Although ADE has been found toresult in disease severity all the severe cases are not associatedwith secondary infection nor do all the cases of secondaryinfection progress to DHFDSS [2] In addition to humoralimmunity cross-reactive memory T cells could also playa role in either providing protective immunity or causingimmunopathology [31]
35 Diagnosis and Clinical Management Dengue infectionis usually confirmed by identification of viral genomic RNAantigens or the antibodies it elicits Antigen detection testsbased on NS1 detection have been designed to detect thedengue viral NS1 proteinwhich gets released from the dengueinfected cells and appears early in the bloodstream A 3-in-1test for simultaneous detection of NS1 IgM and IgG is nowavailable ELISA-based serological tests are easy to performand are cost-effective for dengue detection
Up to date there is no antiviral drug available fordengue Treatment is usually based on symptoms and isperformed through medical support For uncomplicatedcases of dengue fever the treatment prescribed is bed restoral rehydration and paracetamol as an antipyretic andanalgesic Patientrsquos health is monitored through variousblood tests from fever day 3 onwards till the conditionimproves Clinical signs that signal progression to seriousdisease include cold limb extremities low pulse low urineoutput signs of mucosal bleeding and abdominal pain DHFis indicated by a rising hematocrit (ge20) and a fallingplatelet count (gt100000mm3) If any of these signs aredetected immediate hospitalization is necessary Treatmentfor DHF patients is based on intravenous fluid therapy tomaintain effective circulation during plasma leakage pluscareful clinical monitoring of hematocrit platelet countpulse rate and blood pressure temperature urine outputfluid administered and other signs of shock Patients usuallyrecover within 12ndash48 h of fluid therapy Treatment for DSSpatients mainly consists of immediate fluid therapy with
Figure 5 Classification of dengue vaccine candidates
colloids and extensive monitoring of any complicationsIn worse case such as internal hemorrhage whole bloodtransfusion may be carried out [27]
4 Dengue Vaccine Strategies
Despite the existing challenges for an ideal dengue vaccinedevelopment of dengue vaccine candidates has progressedover the last decade and some of these have entered clinicaltrials in both endemic and nonendemic areas A classificationof the current approaches for dengue vaccine development isshown in Figure 5
41 Replicating Viral Vaccines These include live-attenuatedviruses (LAV) that are created by reducing the virulence of apathogen without compromising its viability Current meth-ods of producing live-attenuated viruses for dengue vaccinesinclude attenuation by serial passage in cell lines and targetedmutagenesis and by constructing chimeric vaccine viruses
(i) Advantages robust lasting and broad immunity andlower production cost
(ii) Disadvantages difficulty in attenuation genetic insta-bility possibility of reversion and interference in thecase of multicomponent LAV vaccines
411 Cell Culture Passage Based LAV Development of LAVby serial passage in cell lines was started at Mahidol Univer-sity Bangkok Thailand A tetravalent formulation was madeby attenuating all fourDENV serotypes but the vaccine failedto elicit a balanced immune response despite modulating theviral concentrations [32 33] Increased frequency of adversereactions like fever rash myalgia and retroorbital painprimarily related to theDENV-3 vaccine strain was observedFurther development of these LAV strains was stalled [34]
Another LAV based on passaging in cell culture wasdeveloped by Walter Reed Army Institute of Research(WRAIR) Maryland USA and is being evaluated in clinical
trials in collaboration with GlaxoSmithKline (GSK) Allfour DENV serotypes were attenuated by passaging in pri-mary dog kidney (PDK) cells and a tetravalent formulation(F17Pre) was developed which was found to result in DENV-4 vaccine-induced viremia during phase II clinical trials[35 36] In a separate phase II randomized observer-blindplacebo-controlled trial in 86 healthy flavivirus naıve adultsin USA F17Pre DENVs were rederived and passaged infetus rhesus lung cells to obtain seed viruses of higher purityResultant formulations F17 and F19 containing equivalentamounts of vaccine components except DENV-4 being 10-fold higher in F17 were evaluated An acceptable safetyand immunogenicity profile was observed after 2 doses ofLAV with tetravalent antibody rates of 60 and 67 inparticipants receiving F17 and F19 respectively It was alsoreported that although F19 was formulated to contain 10-foldless DENV-4 it was found to be only fourfold less at the timeof vaccine release The neutralization titers against DENV-4 were found to be comparable at 70 and 46 for F17 andF19 respectively Notably the incidence of DENV-4 vaccine-induced viremia reduced (with only one case in F17 group)probably due to rederivation and passage which attenuatedthe DENV-4 strain further [37] In a similar phase II trialin healthy children and adults in Puerto Rico F17 and F19were evaluated againTheDENV-4 in vitropotency in F19wasfound to be 50-fold less instead of being 10-fold according toformulation design Thus there have been issues related tostorage stability of DENV-4 strain [38]
412 Targeted Mutagenesis Based Live-Attenuated VaccineThis strategy was first successfully explored by the Laboratoryof Infectious Disease at the National Institute of Allergy andInfectious Disease (NIAID) National Institutes of Health(NIH) Maryland USA NIH has established nonexclusivelicense with manufacturers in Brazil (Instituto Butantan)Vietnam (Vabiotech) and India (Serum Institute of Indiaand Panacea Biotech) for its development This vaccinecandidate is amixture of fourDENV strains attenuated by site
Journal of Immunology Research 7
directed mutagenesis to delete 30 nucleotides in the 31015840UTRDENV-1 and DENV-4 attenuated strains were designatedas DEN1Δ30 and DEN4Δ30 respectively [39] DENV-2 andDENV-3 attenuated strains were made by using DEN4Δ30as a backbone and replacing their structural prM and Egenes with those of the corresponding serotype Notablychimerization resulted in overattenuation of rDEN24Δ30and rDEN34Δ30 strainsTheDENV-3 component wasmod-ified variably and rDEN3Δ3031 strain was selected whereadditional 31 nucleotides were deleted from rDEN3Δ30Infectivity of DENV-2 component has been improved intetravalent formulation TV005 by using DENV-2 attenuatedrDEN24Δ30 strain at a 10-fold higher dose (104 pfu) thanother components (103 pfu) tetravalent formulation TV003contains 103 pfu of each of the four components Importantlya single dose of TV005 has been found to be efficacious inproviding sterilizing immunity Additionally TV003TV005are being evaluated in a human challenge model to enable amore stringent assessment of its protective efficacy TV003has been found to protect vaccinees against challenge withDENV-2 attenuated rDEN2Δ30 strain [40 41] Similar evalu-ation of protective efficacy is ongoing for TV005 andDENV-3human challenge experiments are being planned [40] PhaseIII of this vaccine candidate has begun in Brazil [42]
413 Chimeric Dengue Vaccine Chimeric dengue vaccineshave been designed using two approaches (i) with anotherattenuated flavivirus and (ii) with an attenuated DENV strain(intertypic chimera) The vaccine where chimera of DENVhas been made with another flavivirus is the chimeric yellowfever-dengue (CYD) vaccine which is being developed bySanofi Pasteur and licensed under the brand name ldquoDeng-vaxiardquo [4] In this vaccine prM and E genes of the attenuatedyellow fever LAV strain 17D have been replaced with thecorresponding genes from DENV [43] The rationale behindthis design was the fact that humoral response against thestructural proteins of dengue was responsible for protectiveimmunity during natural infection and thus these chimeraswould generate a protective immune response in vaccineesA tetravalent mixture of the four chimeric viruses hasundergone extensive clinical evaluation and has recently beenapproved in Mexico Brazil El Salvador and Philippines[4 44] This vaccine will be discussed in detail in the latersections
An example of intertypic chimera is DENVax developedby Inviragen Inc Fort Collins CO USA DENV-2 strainattenuated by 53 passages in PDK cells (made at MahidolUniversity) has been used as a backbone for generatingchimera prMandE gene of this strainwere replaced by corre-sponding genes fromDENV-1DENV-3 andDENV-4As themutations in the attenuated strain were in the nonstructuralproteins this strain was used as such for DENV-2 componentin the tetravalent formulationThese chimeric viruses showeda temperature-sensitive phenotype reduced replication inmosquito cell lines high degree of genetic stability and lackof neurovirulence in sucklingmice [45]Three tetravalent for-mulations with variable dose of each component were evalu-ated in nonhuman primates It was observed that DENV-2
was the dominating component and its replicative potentialreduced by increasing the DENV-3 andDENV-4 componentThis variation in DENV-2 induced viremia due to the varia-tion in the dose of DENV-3 and DENV-4 components indi-cated viral interference Moreover the neutralizing antibodytiters were found to be significantly low against DENV-4 anddespite this macaques were found to be protected againstDENV-4 challenge [46] A phase I clinical trial of low andhigh doses of the DENVax in healthy subjects in Columbiarevealed that the candidate was safe and immunogenicNotably it corroborated the findings made in nonhuman pri-mate study that neutralizing antibody titers elicited by DEN-Vax are lowest against DENV-4 and highest against DENV-2[47] Further insights into its efficacy will be revealed throughits phase II clinical trial evaluation Meanwhile phase IIIevaluation of this vaccine has now been initiated [48]
42 Nonreplicating Viral Vaccines These vaccine candidatesare not capable of replicating and thus offer the advantage ofconferring immunity without the risk of infection There aremultiple strategies to develop this class of vaccines like DNAvaccines subunit proteins VLPs and so forth
(i) Advantages reduced reactogenicity better suitabilityfor immune-compromised individuals and balancedimmune response in case of tetravalent formulation
(ii) Disadvantages less broad potent and durable im-mune response whichmay result in ADE and requir-ing the use of adjuvants
421 Purified Inactivated Virus (PIV) WRAIR MarylandUSA developed an inactivated monovalent dengue vaccineby formalin treatment It was found to be safe and immuno-genic in mice and Rhesus macaques [49 50] Althoughsuch vaccines would not show viral interference or revertto a pathogenic strain their use as the sole immunizationapproach is limited because of conformational changes invirus by formalin treatment and lack of replication Howeverthis vaccine has been tested as the priming vaccine in a prime-boost immunization strategy with a LAV as the boostervaccine leading to complete protection in macaques [51]Phase I trial evaluating the safety of 25 and 5 120583g of DENV-1component administered on days 0 and 28 in flavivirus naıvepopulation in the USA has been completed [52] Two phase Itrials evaluating tetravalent mixture of the four PIVs (TPIV1 120583g of each of the four PIVs) are being evaluated with alumand two proprietory adjuvants of GSK (AS01E1 and AS03B1)in healthy adults in the USA [53] and in Puerto Rico [54]Healthy adults in the USA are also being recruited in anotherphase I study where TPIValum is being evaluated in prime-boot vaccination with WRAIRGSKrsquos tetravalent LAV [55]
422 Recombinant Subunit Vaccine Recombinant E proteinsof dengue have been expressed in yeast and insect expres-sion systems and have been analyzed for vaccine efficacyin mice and monkeys All these studies have focused onthe DENV E aminoterminal 80 of the molecule knownas the ectodomain Deletion of 20 of the E protein at
8 Journal of Immunology Research
the C-terminal which is a transmembrane region allowsextracellular secretion and easy purification while retainingits antigenicityThe recombinant 80 E proteins also knownas r80E of the four DENV serotypes are being manufac-tured by Hawaii Biotech Inc HI USA and Merck andCo NJ USA Monovalent DEN2 80E was evaluated witha panel of adjuvants in mice and saponin-based adjuvantISCOMATRIXtrade was found to be the most immunogenicimmunogenicity with alum as adjuvant was poor Thiswas followed by evaluation of tetravalent formulation withISCOMATRIX in macaques where titers against DENV-4were found to be the weakest [56] To overcome the lowimmunogenicity of DEN4 80E its dimeric form and doubledose were explored in macaques which led to comparableimprovement in the neutralizing titers against DENV-4Though the titers against DENV-4 improved they were lowerthan the titers against DENV-1 DENV-2 and DENV-3 [57]Based on these results a tetravalent mixture of the four r80Escontaining 10 10 10 and 20120583g of DEN1 DEN2 DEN3and DEN4 80E respectively was further evaluated in flavi-naıve and dengue-primed macaques where it was found togenerate a more balanced immune response against the fourserotypes in 0- 1- and 6-month immunization schedule ascompared to 0 1 and 2 months Moreover two doses (10and 50 120583g) of DEN1 80Ealum administered in flavi-naıveadults on 0 1 and 2 months were found to be safe Howeverit elicited only modest DENV-1 neutralizing titers whichwaned almost completely 26 weeks after the final dose [58]A phase I study examining safety and immunogenicity oftetravalent formulation with and without adjuvant (alum andISCOMATRIX) in healthy adults has been completed [59]
Recombinant antigens based on DENV EDIII have beenproduced by different groups using E coli and yeast expres-sion hosts Recombinant EDIII antigens expressed eitherindependently or fused to different carriers such as maltose-binding protein [60] and the Neisseria meningitides p64kprotein have been shown to generate anti-DENV immuneresponses in mice and nonhuman primates [61ndash63] Thesevaccines candidates are in preclinical phase currently
423 Dengue DNA Vaccine This vaccine consists of a plas-mid vector containing the gene(s) encoding for an antigenwhich on immunization is taken up by antigen presentingcells (APCs) Once the plasmid enters the cell it codes forthe antigen which finally gets associated with MHC classI molecules and gets displayed on the cell surface induc-ing protective cytotoxic immune response Naval MedicalResearch Center (NMRC) USA has developed a DENV-1DNA vaccine candidate (D1ME100) by cloning prM and Egene of DENV-1 serotype into plasmid vector which wasextensively evaluated inmice andmacaques without adjuvant[64] before phase 1 trials in healthy adults AlthoughDENV-1DNA vaccine was found to be well tolerated the neutralizingantibody titers and the number of responders were foundto be low [64 65] Thus to enhance its immunogenicitya lipid-based adjuvant Vaxfectin was explored Tetravalentdengue DNA vaccine (TVDV) was evaluated for immuno-genicity with and without Vaxfectin in macaques It wasobserved that Vaxfectin resulted in higher and more stable
(evaluated till 6 months after final boost) titers The averageneutralization titers with TVDVVaxfectin against DENV-1DENV-2 DENV-3 and DENV-4 a month after final boostwere approximately 200 270 170 and 70 respectively Sixmonths after the final boost the titers against DENV-2 andDENV-3 reduced while those against DENV-1 and DENV-4increased marginally In the group without Vaxfectin titersagainst DENV-2 only were detectable 6 months after the finalboost Moreover Vaxfectin allowed better protection fromviremia against DENV-2 challenge [66] After establishingnontoxicity of TVDVVaxfectin in New Zealand white rab-bits [67] a phase I trial was initiated in 2011 in USA [68]
424 Replication-Defective Virus Vectored Vaccines In thisapproach a virus is used as a vector to carry antigenic genesthat are capable of eliciting neutralizing antibody responseSome examples of viral vectors are adenovirus vectors Ven-ezuelan equine encephalitis virus vector and attenuatedmeasles virus [69ndash71] An example of virus vectored denguevaccine is cAdVax It consists of bivalent constructs express-ing prM and E proteins from two dengue serotypes each(DENV-1 and DENV-3 together in one and DENV-2 andDENV-4 in another construct) Study in NHPs showedproduction of neutralizing antibodies to respective DENVserotypes [72 73] Therefore a tetravalent formulation (cAd-Vax-DenTV) was prepared bymixing the bivalent constructswhich showed protection against all serotypes on DENVchallenge in Rhesus macaques [69]
425 Virus Like Particle (VLP) Vaccines The prM and Eproteins of DENVs coexpressed in heterologous hosts havebeen shown to coassemble into VLPs Thus a vaccine basedon physical mixtures of four monovalent DENV VLPs canbe developed to have a tetravalent formulation [74] Fromthe perspective of using VLPs for vaccine purpose the yeastsystemmay bemore suitable as it has the potential for higheryields and can glycosylate the antigens Recent work indicatesthat yeast expressed-DENV E ectodomain forms VLPs in theabsence of prM [75] Another approach is based on displayingthe DENV EDIII on VLPs formed by hepatitis B virus coreantigens [27]
5 Dengvaxia
Dengue vaccine candidates which have reached clinical trialsare given in Table 2 It is worthwhile to discuss the front-runner CYD vaccine developed by Sanofi Pasteur whichhas recently been approved as Dengvaxia in Mexico BrazilEl Salvador and Philippines [4] Dengvaxia is a tetravalentdengue chimeric live-attenuated virus vaccine based onlicensed yellow fever vaccine 17D It was constructed byreplacement of structural genes of live-attenuated yellowfever virus vaccine 17D with structural genes from eachDENV serotype [76]
51 Preclinical Data The immunogenicity of various tetrava-lent formulations of the chimeric viruses was evaluated inmacaques which revealed immunodominance of serotype
Journal of Immunology Research 9
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
[1] R Chen and N Vasilakis ldquoDengue-Quo Tu et Quo VadisrdquoViruses vol 3 no 9 pp 1562ndash1608 2011
[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
Figure 5 Classification of dengue vaccine candidates
colloids and extensive monitoring of any complicationsIn worse case such as internal hemorrhage whole bloodtransfusion may be carried out [27]
4 Dengue Vaccine Strategies
Despite the existing challenges for an ideal dengue vaccinedevelopment of dengue vaccine candidates has progressedover the last decade and some of these have entered clinicaltrials in both endemic and nonendemic areas A classificationof the current approaches for dengue vaccine development isshown in Figure 5
41 Replicating Viral Vaccines These include live-attenuatedviruses (LAV) that are created by reducing the virulence of apathogen without compromising its viability Current meth-ods of producing live-attenuated viruses for dengue vaccinesinclude attenuation by serial passage in cell lines and targetedmutagenesis and by constructing chimeric vaccine viruses
(i) Advantages robust lasting and broad immunity andlower production cost
(ii) Disadvantages difficulty in attenuation genetic insta-bility possibility of reversion and interference in thecase of multicomponent LAV vaccines
411 Cell Culture Passage Based LAV Development of LAVby serial passage in cell lines was started at Mahidol Univer-sity Bangkok Thailand A tetravalent formulation was madeby attenuating all fourDENV serotypes but the vaccine failedto elicit a balanced immune response despite modulating theviral concentrations [32 33] Increased frequency of adversereactions like fever rash myalgia and retroorbital painprimarily related to theDENV-3 vaccine strain was observedFurther development of these LAV strains was stalled [34]
Another LAV based on passaging in cell culture wasdeveloped by Walter Reed Army Institute of Research(WRAIR) Maryland USA and is being evaluated in clinical
trials in collaboration with GlaxoSmithKline (GSK) Allfour DENV serotypes were attenuated by passaging in pri-mary dog kidney (PDK) cells and a tetravalent formulation(F17Pre) was developed which was found to result in DENV-4 vaccine-induced viremia during phase II clinical trials[35 36] In a separate phase II randomized observer-blindplacebo-controlled trial in 86 healthy flavivirus naıve adultsin USA F17Pre DENVs were rederived and passaged infetus rhesus lung cells to obtain seed viruses of higher purityResultant formulations F17 and F19 containing equivalentamounts of vaccine components except DENV-4 being 10-fold higher in F17 were evaluated An acceptable safetyand immunogenicity profile was observed after 2 doses ofLAV with tetravalent antibody rates of 60 and 67 inparticipants receiving F17 and F19 respectively It was alsoreported that although F19 was formulated to contain 10-foldless DENV-4 it was found to be only fourfold less at the timeof vaccine release The neutralization titers against DENV-4 were found to be comparable at 70 and 46 for F17 andF19 respectively Notably the incidence of DENV-4 vaccine-induced viremia reduced (with only one case in F17 group)probably due to rederivation and passage which attenuatedthe DENV-4 strain further [37] In a similar phase II trialin healthy children and adults in Puerto Rico F17 and F19were evaluated againTheDENV-4 in vitropotency in F19wasfound to be 50-fold less instead of being 10-fold according toformulation design Thus there have been issues related tostorage stability of DENV-4 strain [38]
412 Targeted Mutagenesis Based Live-Attenuated VaccineThis strategy was first successfully explored by the Laboratoryof Infectious Disease at the National Institute of Allergy andInfectious Disease (NIAID) National Institutes of Health(NIH) Maryland USA NIH has established nonexclusivelicense with manufacturers in Brazil (Instituto Butantan)Vietnam (Vabiotech) and India (Serum Institute of Indiaand Panacea Biotech) for its development This vaccinecandidate is amixture of fourDENV strains attenuated by site
Journal of Immunology Research 7
directed mutagenesis to delete 30 nucleotides in the 31015840UTRDENV-1 and DENV-4 attenuated strains were designatedas DEN1Δ30 and DEN4Δ30 respectively [39] DENV-2 andDENV-3 attenuated strains were made by using DEN4Δ30as a backbone and replacing their structural prM and Egenes with those of the corresponding serotype Notablychimerization resulted in overattenuation of rDEN24Δ30and rDEN34Δ30 strainsTheDENV-3 component wasmod-ified variably and rDEN3Δ3031 strain was selected whereadditional 31 nucleotides were deleted from rDEN3Δ30Infectivity of DENV-2 component has been improved intetravalent formulation TV005 by using DENV-2 attenuatedrDEN24Δ30 strain at a 10-fold higher dose (104 pfu) thanother components (103 pfu) tetravalent formulation TV003contains 103 pfu of each of the four components Importantlya single dose of TV005 has been found to be efficacious inproviding sterilizing immunity Additionally TV003TV005are being evaluated in a human challenge model to enable amore stringent assessment of its protective efficacy TV003has been found to protect vaccinees against challenge withDENV-2 attenuated rDEN2Δ30 strain [40 41] Similar evalu-ation of protective efficacy is ongoing for TV005 andDENV-3human challenge experiments are being planned [40] PhaseIII of this vaccine candidate has begun in Brazil [42]
413 Chimeric Dengue Vaccine Chimeric dengue vaccineshave been designed using two approaches (i) with anotherattenuated flavivirus and (ii) with an attenuated DENV strain(intertypic chimera) The vaccine where chimera of DENVhas been made with another flavivirus is the chimeric yellowfever-dengue (CYD) vaccine which is being developed bySanofi Pasteur and licensed under the brand name ldquoDeng-vaxiardquo [4] In this vaccine prM and E genes of the attenuatedyellow fever LAV strain 17D have been replaced with thecorresponding genes from DENV [43] The rationale behindthis design was the fact that humoral response against thestructural proteins of dengue was responsible for protectiveimmunity during natural infection and thus these chimeraswould generate a protective immune response in vaccineesA tetravalent mixture of the four chimeric viruses hasundergone extensive clinical evaluation and has recently beenapproved in Mexico Brazil El Salvador and Philippines[4 44] This vaccine will be discussed in detail in the latersections
An example of intertypic chimera is DENVax developedby Inviragen Inc Fort Collins CO USA DENV-2 strainattenuated by 53 passages in PDK cells (made at MahidolUniversity) has been used as a backbone for generatingchimera prMandE gene of this strainwere replaced by corre-sponding genes fromDENV-1DENV-3 andDENV-4As themutations in the attenuated strain were in the nonstructuralproteins this strain was used as such for DENV-2 componentin the tetravalent formulationThese chimeric viruses showeda temperature-sensitive phenotype reduced replication inmosquito cell lines high degree of genetic stability and lackof neurovirulence in sucklingmice [45]Three tetravalent for-mulations with variable dose of each component were evalu-ated in nonhuman primates It was observed that DENV-2
was the dominating component and its replicative potentialreduced by increasing the DENV-3 andDENV-4 componentThis variation in DENV-2 induced viremia due to the varia-tion in the dose of DENV-3 and DENV-4 components indi-cated viral interference Moreover the neutralizing antibodytiters were found to be significantly low against DENV-4 anddespite this macaques were found to be protected againstDENV-4 challenge [46] A phase I clinical trial of low andhigh doses of the DENVax in healthy subjects in Columbiarevealed that the candidate was safe and immunogenicNotably it corroborated the findings made in nonhuman pri-mate study that neutralizing antibody titers elicited by DEN-Vax are lowest against DENV-4 and highest against DENV-2[47] Further insights into its efficacy will be revealed throughits phase II clinical trial evaluation Meanwhile phase IIIevaluation of this vaccine has now been initiated [48]
42 Nonreplicating Viral Vaccines These vaccine candidatesare not capable of replicating and thus offer the advantage ofconferring immunity without the risk of infection There aremultiple strategies to develop this class of vaccines like DNAvaccines subunit proteins VLPs and so forth
(i) Advantages reduced reactogenicity better suitabilityfor immune-compromised individuals and balancedimmune response in case of tetravalent formulation
(ii) Disadvantages less broad potent and durable im-mune response whichmay result in ADE and requir-ing the use of adjuvants
421 Purified Inactivated Virus (PIV) WRAIR MarylandUSA developed an inactivated monovalent dengue vaccineby formalin treatment It was found to be safe and immuno-genic in mice and Rhesus macaques [49 50] Althoughsuch vaccines would not show viral interference or revertto a pathogenic strain their use as the sole immunizationapproach is limited because of conformational changes invirus by formalin treatment and lack of replication Howeverthis vaccine has been tested as the priming vaccine in a prime-boost immunization strategy with a LAV as the boostervaccine leading to complete protection in macaques [51]Phase I trial evaluating the safety of 25 and 5 120583g of DENV-1component administered on days 0 and 28 in flavivirus naıvepopulation in the USA has been completed [52] Two phase Itrials evaluating tetravalent mixture of the four PIVs (TPIV1 120583g of each of the four PIVs) are being evaluated with alumand two proprietory adjuvants of GSK (AS01E1 and AS03B1)in healthy adults in the USA [53] and in Puerto Rico [54]Healthy adults in the USA are also being recruited in anotherphase I study where TPIValum is being evaluated in prime-boot vaccination with WRAIRGSKrsquos tetravalent LAV [55]
422 Recombinant Subunit Vaccine Recombinant E proteinsof dengue have been expressed in yeast and insect expres-sion systems and have been analyzed for vaccine efficacyin mice and monkeys All these studies have focused onthe DENV E aminoterminal 80 of the molecule knownas the ectodomain Deletion of 20 of the E protein at
8 Journal of Immunology Research
the C-terminal which is a transmembrane region allowsextracellular secretion and easy purification while retainingits antigenicityThe recombinant 80 E proteins also knownas r80E of the four DENV serotypes are being manufac-tured by Hawaii Biotech Inc HI USA and Merck andCo NJ USA Monovalent DEN2 80E was evaluated witha panel of adjuvants in mice and saponin-based adjuvantISCOMATRIXtrade was found to be the most immunogenicimmunogenicity with alum as adjuvant was poor Thiswas followed by evaluation of tetravalent formulation withISCOMATRIX in macaques where titers against DENV-4were found to be the weakest [56] To overcome the lowimmunogenicity of DEN4 80E its dimeric form and doubledose were explored in macaques which led to comparableimprovement in the neutralizing titers against DENV-4Though the titers against DENV-4 improved they were lowerthan the titers against DENV-1 DENV-2 and DENV-3 [57]Based on these results a tetravalent mixture of the four r80Escontaining 10 10 10 and 20120583g of DEN1 DEN2 DEN3and DEN4 80E respectively was further evaluated in flavi-naıve and dengue-primed macaques where it was found togenerate a more balanced immune response against the fourserotypes in 0- 1- and 6-month immunization schedule ascompared to 0 1 and 2 months Moreover two doses (10and 50 120583g) of DEN1 80Ealum administered in flavi-naıveadults on 0 1 and 2 months were found to be safe Howeverit elicited only modest DENV-1 neutralizing titers whichwaned almost completely 26 weeks after the final dose [58]A phase I study examining safety and immunogenicity oftetravalent formulation with and without adjuvant (alum andISCOMATRIX) in healthy adults has been completed [59]
Recombinant antigens based on DENV EDIII have beenproduced by different groups using E coli and yeast expres-sion hosts Recombinant EDIII antigens expressed eitherindependently or fused to different carriers such as maltose-binding protein [60] and the Neisseria meningitides p64kprotein have been shown to generate anti-DENV immuneresponses in mice and nonhuman primates [61ndash63] Thesevaccines candidates are in preclinical phase currently
423 Dengue DNA Vaccine This vaccine consists of a plas-mid vector containing the gene(s) encoding for an antigenwhich on immunization is taken up by antigen presentingcells (APCs) Once the plasmid enters the cell it codes forthe antigen which finally gets associated with MHC classI molecules and gets displayed on the cell surface induc-ing protective cytotoxic immune response Naval MedicalResearch Center (NMRC) USA has developed a DENV-1DNA vaccine candidate (D1ME100) by cloning prM and Egene of DENV-1 serotype into plasmid vector which wasextensively evaluated inmice andmacaques without adjuvant[64] before phase 1 trials in healthy adults AlthoughDENV-1DNA vaccine was found to be well tolerated the neutralizingantibody titers and the number of responders were foundto be low [64 65] Thus to enhance its immunogenicitya lipid-based adjuvant Vaxfectin was explored Tetravalentdengue DNA vaccine (TVDV) was evaluated for immuno-genicity with and without Vaxfectin in macaques It wasobserved that Vaxfectin resulted in higher and more stable
(evaluated till 6 months after final boost) titers The averageneutralization titers with TVDVVaxfectin against DENV-1DENV-2 DENV-3 and DENV-4 a month after final boostwere approximately 200 270 170 and 70 respectively Sixmonths after the final boost the titers against DENV-2 andDENV-3 reduced while those against DENV-1 and DENV-4increased marginally In the group without Vaxfectin titersagainst DENV-2 only were detectable 6 months after the finalboost Moreover Vaxfectin allowed better protection fromviremia against DENV-2 challenge [66] After establishingnontoxicity of TVDVVaxfectin in New Zealand white rab-bits [67] a phase I trial was initiated in 2011 in USA [68]
424 Replication-Defective Virus Vectored Vaccines In thisapproach a virus is used as a vector to carry antigenic genesthat are capable of eliciting neutralizing antibody responseSome examples of viral vectors are adenovirus vectors Ven-ezuelan equine encephalitis virus vector and attenuatedmeasles virus [69ndash71] An example of virus vectored denguevaccine is cAdVax It consists of bivalent constructs express-ing prM and E proteins from two dengue serotypes each(DENV-1 and DENV-3 together in one and DENV-2 andDENV-4 in another construct) Study in NHPs showedproduction of neutralizing antibodies to respective DENVserotypes [72 73] Therefore a tetravalent formulation (cAd-Vax-DenTV) was prepared bymixing the bivalent constructswhich showed protection against all serotypes on DENVchallenge in Rhesus macaques [69]
425 Virus Like Particle (VLP) Vaccines The prM and Eproteins of DENVs coexpressed in heterologous hosts havebeen shown to coassemble into VLPs Thus a vaccine basedon physical mixtures of four monovalent DENV VLPs canbe developed to have a tetravalent formulation [74] Fromthe perspective of using VLPs for vaccine purpose the yeastsystemmay bemore suitable as it has the potential for higheryields and can glycosylate the antigens Recent work indicatesthat yeast expressed-DENV E ectodomain forms VLPs in theabsence of prM [75] Another approach is based on displayingthe DENV EDIII on VLPs formed by hepatitis B virus coreantigens [27]
5 Dengvaxia
Dengue vaccine candidates which have reached clinical trialsare given in Table 2 It is worthwhile to discuss the front-runner CYD vaccine developed by Sanofi Pasteur whichhas recently been approved as Dengvaxia in Mexico BrazilEl Salvador and Philippines [4] Dengvaxia is a tetravalentdengue chimeric live-attenuated virus vaccine based onlicensed yellow fever vaccine 17D It was constructed byreplacement of structural genes of live-attenuated yellowfever virus vaccine 17D with structural genes from eachDENV serotype [76]
51 Preclinical Data The immunogenicity of various tetrava-lent formulations of the chimeric viruses was evaluated inmacaques which revealed immunodominance of serotype
Journal of Immunology Research 9
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
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[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
directed mutagenesis to delete 30 nucleotides in the 31015840UTRDENV-1 and DENV-4 attenuated strains were designatedas DEN1Δ30 and DEN4Δ30 respectively [39] DENV-2 andDENV-3 attenuated strains were made by using DEN4Δ30as a backbone and replacing their structural prM and Egenes with those of the corresponding serotype Notablychimerization resulted in overattenuation of rDEN24Δ30and rDEN34Δ30 strainsTheDENV-3 component wasmod-ified variably and rDEN3Δ3031 strain was selected whereadditional 31 nucleotides were deleted from rDEN3Δ30Infectivity of DENV-2 component has been improved intetravalent formulation TV005 by using DENV-2 attenuatedrDEN24Δ30 strain at a 10-fold higher dose (104 pfu) thanother components (103 pfu) tetravalent formulation TV003contains 103 pfu of each of the four components Importantlya single dose of TV005 has been found to be efficacious inproviding sterilizing immunity Additionally TV003TV005are being evaluated in a human challenge model to enable amore stringent assessment of its protective efficacy TV003has been found to protect vaccinees against challenge withDENV-2 attenuated rDEN2Δ30 strain [40 41] Similar evalu-ation of protective efficacy is ongoing for TV005 andDENV-3human challenge experiments are being planned [40] PhaseIII of this vaccine candidate has begun in Brazil [42]
413 Chimeric Dengue Vaccine Chimeric dengue vaccineshave been designed using two approaches (i) with anotherattenuated flavivirus and (ii) with an attenuated DENV strain(intertypic chimera) The vaccine where chimera of DENVhas been made with another flavivirus is the chimeric yellowfever-dengue (CYD) vaccine which is being developed bySanofi Pasteur and licensed under the brand name ldquoDeng-vaxiardquo [4] In this vaccine prM and E genes of the attenuatedyellow fever LAV strain 17D have been replaced with thecorresponding genes from DENV [43] The rationale behindthis design was the fact that humoral response against thestructural proteins of dengue was responsible for protectiveimmunity during natural infection and thus these chimeraswould generate a protective immune response in vaccineesA tetravalent mixture of the four chimeric viruses hasundergone extensive clinical evaluation and has recently beenapproved in Mexico Brazil El Salvador and Philippines[4 44] This vaccine will be discussed in detail in the latersections
An example of intertypic chimera is DENVax developedby Inviragen Inc Fort Collins CO USA DENV-2 strainattenuated by 53 passages in PDK cells (made at MahidolUniversity) has been used as a backbone for generatingchimera prMandE gene of this strainwere replaced by corre-sponding genes fromDENV-1DENV-3 andDENV-4As themutations in the attenuated strain were in the nonstructuralproteins this strain was used as such for DENV-2 componentin the tetravalent formulationThese chimeric viruses showeda temperature-sensitive phenotype reduced replication inmosquito cell lines high degree of genetic stability and lackof neurovirulence in sucklingmice [45]Three tetravalent for-mulations with variable dose of each component were evalu-ated in nonhuman primates It was observed that DENV-2
was the dominating component and its replicative potentialreduced by increasing the DENV-3 andDENV-4 componentThis variation in DENV-2 induced viremia due to the varia-tion in the dose of DENV-3 and DENV-4 components indi-cated viral interference Moreover the neutralizing antibodytiters were found to be significantly low against DENV-4 anddespite this macaques were found to be protected againstDENV-4 challenge [46] A phase I clinical trial of low andhigh doses of the DENVax in healthy subjects in Columbiarevealed that the candidate was safe and immunogenicNotably it corroborated the findings made in nonhuman pri-mate study that neutralizing antibody titers elicited by DEN-Vax are lowest against DENV-4 and highest against DENV-2[47] Further insights into its efficacy will be revealed throughits phase II clinical trial evaluation Meanwhile phase IIIevaluation of this vaccine has now been initiated [48]
42 Nonreplicating Viral Vaccines These vaccine candidatesare not capable of replicating and thus offer the advantage ofconferring immunity without the risk of infection There aremultiple strategies to develop this class of vaccines like DNAvaccines subunit proteins VLPs and so forth
(i) Advantages reduced reactogenicity better suitabilityfor immune-compromised individuals and balancedimmune response in case of tetravalent formulation
(ii) Disadvantages less broad potent and durable im-mune response whichmay result in ADE and requir-ing the use of adjuvants
421 Purified Inactivated Virus (PIV) WRAIR MarylandUSA developed an inactivated monovalent dengue vaccineby formalin treatment It was found to be safe and immuno-genic in mice and Rhesus macaques [49 50] Althoughsuch vaccines would not show viral interference or revertto a pathogenic strain their use as the sole immunizationapproach is limited because of conformational changes invirus by formalin treatment and lack of replication Howeverthis vaccine has been tested as the priming vaccine in a prime-boost immunization strategy with a LAV as the boostervaccine leading to complete protection in macaques [51]Phase I trial evaluating the safety of 25 and 5 120583g of DENV-1component administered on days 0 and 28 in flavivirus naıvepopulation in the USA has been completed [52] Two phase Itrials evaluating tetravalent mixture of the four PIVs (TPIV1 120583g of each of the four PIVs) are being evaluated with alumand two proprietory adjuvants of GSK (AS01E1 and AS03B1)in healthy adults in the USA [53] and in Puerto Rico [54]Healthy adults in the USA are also being recruited in anotherphase I study where TPIValum is being evaluated in prime-boot vaccination with WRAIRGSKrsquos tetravalent LAV [55]
422 Recombinant Subunit Vaccine Recombinant E proteinsof dengue have been expressed in yeast and insect expres-sion systems and have been analyzed for vaccine efficacyin mice and monkeys All these studies have focused onthe DENV E aminoterminal 80 of the molecule knownas the ectodomain Deletion of 20 of the E protein at
8 Journal of Immunology Research
the C-terminal which is a transmembrane region allowsextracellular secretion and easy purification while retainingits antigenicityThe recombinant 80 E proteins also knownas r80E of the four DENV serotypes are being manufac-tured by Hawaii Biotech Inc HI USA and Merck andCo NJ USA Monovalent DEN2 80E was evaluated witha panel of adjuvants in mice and saponin-based adjuvantISCOMATRIXtrade was found to be the most immunogenicimmunogenicity with alum as adjuvant was poor Thiswas followed by evaluation of tetravalent formulation withISCOMATRIX in macaques where titers against DENV-4were found to be the weakest [56] To overcome the lowimmunogenicity of DEN4 80E its dimeric form and doubledose were explored in macaques which led to comparableimprovement in the neutralizing titers against DENV-4Though the titers against DENV-4 improved they were lowerthan the titers against DENV-1 DENV-2 and DENV-3 [57]Based on these results a tetravalent mixture of the four r80Escontaining 10 10 10 and 20120583g of DEN1 DEN2 DEN3and DEN4 80E respectively was further evaluated in flavi-naıve and dengue-primed macaques where it was found togenerate a more balanced immune response against the fourserotypes in 0- 1- and 6-month immunization schedule ascompared to 0 1 and 2 months Moreover two doses (10and 50 120583g) of DEN1 80Ealum administered in flavi-naıveadults on 0 1 and 2 months were found to be safe Howeverit elicited only modest DENV-1 neutralizing titers whichwaned almost completely 26 weeks after the final dose [58]A phase I study examining safety and immunogenicity oftetravalent formulation with and without adjuvant (alum andISCOMATRIX) in healthy adults has been completed [59]
Recombinant antigens based on DENV EDIII have beenproduced by different groups using E coli and yeast expres-sion hosts Recombinant EDIII antigens expressed eitherindependently or fused to different carriers such as maltose-binding protein [60] and the Neisseria meningitides p64kprotein have been shown to generate anti-DENV immuneresponses in mice and nonhuman primates [61ndash63] Thesevaccines candidates are in preclinical phase currently
423 Dengue DNA Vaccine This vaccine consists of a plas-mid vector containing the gene(s) encoding for an antigenwhich on immunization is taken up by antigen presentingcells (APCs) Once the plasmid enters the cell it codes forthe antigen which finally gets associated with MHC classI molecules and gets displayed on the cell surface induc-ing protective cytotoxic immune response Naval MedicalResearch Center (NMRC) USA has developed a DENV-1DNA vaccine candidate (D1ME100) by cloning prM and Egene of DENV-1 serotype into plasmid vector which wasextensively evaluated inmice andmacaques without adjuvant[64] before phase 1 trials in healthy adults AlthoughDENV-1DNA vaccine was found to be well tolerated the neutralizingantibody titers and the number of responders were foundto be low [64 65] Thus to enhance its immunogenicitya lipid-based adjuvant Vaxfectin was explored Tetravalentdengue DNA vaccine (TVDV) was evaluated for immuno-genicity with and without Vaxfectin in macaques It wasobserved that Vaxfectin resulted in higher and more stable
(evaluated till 6 months after final boost) titers The averageneutralization titers with TVDVVaxfectin against DENV-1DENV-2 DENV-3 and DENV-4 a month after final boostwere approximately 200 270 170 and 70 respectively Sixmonths after the final boost the titers against DENV-2 andDENV-3 reduced while those against DENV-1 and DENV-4increased marginally In the group without Vaxfectin titersagainst DENV-2 only were detectable 6 months after the finalboost Moreover Vaxfectin allowed better protection fromviremia against DENV-2 challenge [66] After establishingnontoxicity of TVDVVaxfectin in New Zealand white rab-bits [67] a phase I trial was initiated in 2011 in USA [68]
424 Replication-Defective Virus Vectored Vaccines In thisapproach a virus is used as a vector to carry antigenic genesthat are capable of eliciting neutralizing antibody responseSome examples of viral vectors are adenovirus vectors Ven-ezuelan equine encephalitis virus vector and attenuatedmeasles virus [69ndash71] An example of virus vectored denguevaccine is cAdVax It consists of bivalent constructs express-ing prM and E proteins from two dengue serotypes each(DENV-1 and DENV-3 together in one and DENV-2 andDENV-4 in another construct) Study in NHPs showedproduction of neutralizing antibodies to respective DENVserotypes [72 73] Therefore a tetravalent formulation (cAd-Vax-DenTV) was prepared bymixing the bivalent constructswhich showed protection against all serotypes on DENVchallenge in Rhesus macaques [69]
425 Virus Like Particle (VLP) Vaccines The prM and Eproteins of DENVs coexpressed in heterologous hosts havebeen shown to coassemble into VLPs Thus a vaccine basedon physical mixtures of four monovalent DENV VLPs canbe developed to have a tetravalent formulation [74] Fromthe perspective of using VLPs for vaccine purpose the yeastsystemmay bemore suitable as it has the potential for higheryields and can glycosylate the antigens Recent work indicatesthat yeast expressed-DENV E ectodomain forms VLPs in theabsence of prM [75] Another approach is based on displayingthe DENV EDIII on VLPs formed by hepatitis B virus coreantigens [27]
5 Dengvaxia
Dengue vaccine candidates which have reached clinical trialsare given in Table 2 It is worthwhile to discuss the front-runner CYD vaccine developed by Sanofi Pasteur whichhas recently been approved as Dengvaxia in Mexico BrazilEl Salvador and Philippines [4] Dengvaxia is a tetravalentdengue chimeric live-attenuated virus vaccine based onlicensed yellow fever vaccine 17D It was constructed byreplacement of structural genes of live-attenuated yellowfever virus vaccine 17D with structural genes from eachDENV serotype [76]
51 Preclinical Data The immunogenicity of various tetrava-lent formulations of the chimeric viruses was evaluated inmacaques which revealed immunodominance of serotype
Journal of Immunology Research 9
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
[1] R Chen and N Vasilakis ldquoDengue-Quo Tu et Quo VadisrdquoViruses vol 3 no 9 pp 1562ndash1608 2011
[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
the C-terminal which is a transmembrane region allowsextracellular secretion and easy purification while retainingits antigenicityThe recombinant 80 E proteins also knownas r80E of the four DENV serotypes are being manufac-tured by Hawaii Biotech Inc HI USA and Merck andCo NJ USA Monovalent DEN2 80E was evaluated witha panel of adjuvants in mice and saponin-based adjuvantISCOMATRIXtrade was found to be the most immunogenicimmunogenicity with alum as adjuvant was poor Thiswas followed by evaluation of tetravalent formulation withISCOMATRIX in macaques where titers against DENV-4were found to be the weakest [56] To overcome the lowimmunogenicity of DEN4 80E its dimeric form and doubledose were explored in macaques which led to comparableimprovement in the neutralizing titers against DENV-4Though the titers against DENV-4 improved they were lowerthan the titers against DENV-1 DENV-2 and DENV-3 [57]Based on these results a tetravalent mixture of the four r80Escontaining 10 10 10 and 20120583g of DEN1 DEN2 DEN3and DEN4 80E respectively was further evaluated in flavi-naıve and dengue-primed macaques where it was found togenerate a more balanced immune response against the fourserotypes in 0- 1- and 6-month immunization schedule ascompared to 0 1 and 2 months Moreover two doses (10and 50 120583g) of DEN1 80Ealum administered in flavi-naıveadults on 0 1 and 2 months were found to be safe Howeverit elicited only modest DENV-1 neutralizing titers whichwaned almost completely 26 weeks after the final dose [58]A phase I study examining safety and immunogenicity oftetravalent formulation with and without adjuvant (alum andISCOMATRIX) in healthy adults has been completed [59]
Recombinant antigens based on DENV EDIII have beenproduced by different groups using E coli and yeast expres-sion hosts Recombinant EDIII antigens expressed eitherindependently or fused to different carriers such as maltose-binding protein [60] and the Neisseria meningitides p64kprotein have been shown to generate anti-DENV immuneresponses in mice and nonhuman primates [61ndash63] Thesevaccines candidates are in preclinical phase currently
423 Dengue DNA Vaccine This vaccine consists of a plas-mid vector containing the gene(s) encoding for an antigenwhich on immunization is taken up by antigen presentingcells (APCs) Once the plasmid enters the cell it codes forthe antigen which finally gets associated with MHC classI molecules and gets displayed on the cell surface induc-ing protective cytotoxic immune response Naval MedicalResearch Center (NMRC) USA has developed a DENV-1DNA vaccine candidate (D1ME100) by cloning prM and Egene of DENV-1 serotype into plasmid vector which wasextensively evaluated inmice andmacaques without adjuvant[64] before phase 1 trials in healthy adults AlthoughDENV-1DNA vaccine was found to be well tolerated the neutralizingantibody titers and the number of responders were foundto be low [64 65] Thus to enhance its immunogenicitya lipid-based adjuvant Vaxfectin was explored Tetravalentdengue DNA vaccine (TVDV) was evaluated for immuno-genicity with and without Vaxfectin in macaques It wasobserved that Vaxfectin resulted in higher and more stable
(evaluated till 6 months after final boost) titers The averageneutralization titers with TVDVVaxfectin against DENV-1DENV-2 DENV-3 and DENV-4 a month after final boostwere approximately 200 270 170 and 70 respectively Sixmonths after the final boost the titers against DENV-2 andDENV-3 reduced while those against DENV-1 and DENV-4increased marginally In the group without Vaxfectin titersagainst DENV-2 only were detectable 6 months after the finalboost Moreover Vaxfectin allowed better protection fromviremia against DENV-2 challenge [66] After establishingnontoxicity of TVDVVaxfectin in New Zealand white rab-bits [67] a phase I trial was initiated in 2011 in USA [68]
424 Replication-Defective Virus Vectored Vaccines In thisapproach a virus is used as a vector to carry antigenic genesthat are capable of eliciting neutralizing antibody responseSome examples of viral vectors are adenovirus vectors Ven-ezuelan equine encephalitis virus vector and attenuatedmeasles virus [69ndash71] An example of virus vectored denguevaccine is cAdVax It consists of bivalent constructs express-ing prM and E proteins from two dengue serotypes each(DENV-1 and DENV-3 together in one and DENV-2 andDENV-4 in another construct) Study in NHPs showedproduction of neutralizing antibodies to respective DENVserotypes [72 73] Therefore a tetravalent formulation (cAd-Vax-DenTV) was prepared bymixing the bivalent constructswhich showed protection against all serotypes on DENVchallenge in Rhesus macaques [69]
425 Virus Like Particle (VLP) Vaccines The prM and Eproteins of DENVs coexpressed in heterologous hosts havebeen shown to coassemble into VLPs Thus a vaccine basedon physical mixtures of four monovalent DENV VLPs canbe developed to have a tetravalent formulation [74] Fromthe perspective of using VLPs for vaccine purpose the yeastsystemmay bemore suitable as it has the potential for higheryields and can glycosylate the antigens Recent work indicatesthat yeast expressed-DENV E ectodomain forms VLPs in theabsence of prM [75] Another approach is based on displayingthe DENV EDIII on VLPs formed by hepatitis B virus coreantigens [27]
5 Dengvaxia
Dengue vaccine candidates which have reached clinical trialsare given in Table 2 It is worthwhile to discuss the front-runner CYD vaccine developed by Sanofi Pasteur whichhas recently been approved as Dengvaxia in Mexico BrazilEl Salvador and Philippines [4] Dengvaxia is a tetravalentdengue chimeric live-attenuated virus vaccine based onlicensed yellow fever vaccine 17D It was constructed byreplacement of structural genes of live-attenuated yellowfever virus vaccine 17D with structural genes from eachDENV serotype [76]
51 Preclinical Data The immunogenicity of various tetrava-lent formulations of the chimeric viruses was evaluated inmacaques which revealed immunodominance of serotype
Journal of Immunology Research 9
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
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[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
Table 2 Dengue vaccine candidates currently in different phases ofclinical trials
Type of vaccine Developer PhaseChimeric yellow virusdengue vaccine (CYD) Sanofi Pasteur Licensed
Intertypicchimera-DENVax CDC-InviragenTakeda III
Targeted mutagenesis basedLAV-TetraVax-DV NIH III
Cell culture based LAV WRAIR-GSK IIPurified inactivatedvaccine-TDENV-PIV WRAIR-GSK I
Recombinant subunitvaccine-V180
Hawaii Biotech Merckand Co I
DNA vaccine expressingprM and E protein
Naval Medical ResearchCentre WRAIR I
4 chimeric virus neutralizing antibody titers in the elicitedresponses were consistently lowest against DENV-2 [77 78]and they failed to confer solid protective immunity to wilddengue challenge [79]
52 Phase I Trial Monovalent serotype 2 chimera evaluatedin a phase I study in healthy adults 18ndash49 years old was foundto be safe and immunogenic [80] Therefore tetravalentformulation containing 5 log
10cell culture infective dose 50
(CCID50) was tested in dengue naıve US adults aged 18ndash45years The vaccine was well tolerated with all the participantsseroconverting to all four DENV serotypes after receivingthree doses of the vaccine However low levels of viremiawere observed primarily againstDENV-4 [81] Another phaseI trial was conducted in dengue endemic area like PhilippinesHere the vaccine was evaluated on subjects of four agecohorts 2ndash5 6ndash11 12ndash17 and 18ndash45 years Vaccine was foundto be safe and all the vaccinees exhibited high seroconversionrate (gt88) for all the four DENV serotypes [82] Thusthe tetravalent vaccine was safe and immunogenic in bothdengue endemic and nonendemic areas
53 Phase II and Phase IIb Trials A randomized double-blind multicenter phase II trial was conducted in healthyUS adults to test various tetravalent formulations for theCYD-TDV vaccine Although all vaccine formulations weresafe and immunogenic the formulation containing 5 log
10
tissue culture infective dose 50 (TCID 50) of each serotypedemonstrated the best immunogenicityThis formulationwasused for further studies [83] Another randomized controlledphase IIb trial was conducted in 4ndash11-year-old school childrenat Ratchaburi Province Thailand The overall efficacy ofCYD-TDV was found to be a low 302 (95 CI minus134 to566) after 3 doses Moreover the efficacy was highly variablebetween the various serotypes 556 (95 CI minus216 to 840)for DENV-1 92 (95 CI minus750 to 513) for DENV-2 753(95 CI minus3750 to 996) for DENV-3 and 100 (95 CI 248to 1000) for DENV-4 [84] It should be noted that confidenceintervals of all the efficacies except that against DENV-4
included zero which raises concerns over the significance ofthese results
The lack of efficacy against DENV-2 in this trial may beattributed to the following reasons
(i) The genotype of DENV-2 circulating inThailand hadan antigenic mismatch with the vaccine virus straindue to mutations in E [85]
(ii) PRNT assay used to determine the neutralizing anti-body titers during the trials was carried out in Verocells that lack the Fc120574 receptors on the cell surface AsADE can play an important role in vivo using thesereceptors this assay may not truly predict vaccineefficacy [85]
(iii) As the vaccine molecule contained many cross-reactive epitopes therefore it is possible that en-hancement took over the neutralization potential ofantibodies in vivo leading to poor efficacy as was laterobserved during phase III trials too [86]
Another randomized blinded controlled phase II trial wasconducted in 9ndash16-year-old subjects fromLatinAmericaTheseropositivity for at least two three or all four serotypes was100 906 and 934 respectively after 3 doses Vaccineeswho were seropositive for flavivirus antibodies before immu-nization had higher antibody titers upon immunization (ascompared to seronegative subjects) The rates of virologicallyconfirmed dengue cases for all four DENV serotypes werelower in the vaccine group compared to that in the controlgroup The contrast in results between this trial and the oneconducted in Thailand was attributed to the difference inepidemiology and circulating virus strain differences betweenthe two countries [87]
54 Phase III Trial An observer-masked randomized con-trolled multicenter phase III trial was done on healthychildren aged 2ndash14 years in 5 countries ofAsia-Pacific regionsThey were randomly assigned (stratified by age and site) toreceive three doses of CYD-TDV or placebo at 0 6 and12 months Subjects were followed up until 25 months Theprimary endpoint was achieved with 565 (95 CI 438ndash664) efficacy Thus the vaccine was found to be moderatelyefficacious Though the overall efficacy improved it remainslow and statistically insignificant against DENV-2 at 350(95CIminus92 to 610) [88] A follow-up of the vaccinees in year3 to score the relative risk of hospitalization for virologicallyconfirmed dengue revealed alarming results for childrenbetween 2 and 5 yearsThe rate of hospitalization of vaccineesof this age group was more than seven times the controlgroup Overall the relative risk of hospitalization for childrenlt9 years was 158 as compared to the alarming 745 for 2ndash5-year-old children Moreover vaccine efficacy was also foundto be lower in vaccinees lt9 years of age The overall vaccineefficacy was 678 (95 CI 575 to 756) and 446 (95 CI316 to 550) for participants above and below 9 years of agerespectivelyThis difference in efficacy was more pronouncedin dengue naıve participants where overall efficacy wasreported to be 616 (95 CI minus211 to 881) and a poor 144(95 CI minus111 to 635) in participants above and below 9 years
10 Journal of Immunology Research
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
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[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
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[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
of age respectively [89]The outcome that CYD-TDVvaccineputs children lt9 years of age at greater risk of hospitalizationis a serious safety concern It is believed that CYD-TDVsensitized the dengue naıve subjects of all the age groups(owing to its low efficacy) to enhanced dengue infectionincreasing the risk of hospitalization Although it was foundto be efficacious in reducing the risk of hospitalization inseropositive recipients it has been estimated that for everytwo recipients prevented from hospitalization one recipientwas hospitalized due to vaccine-induced enhanced disease[86] These concerns have put children lt9 years of age anddengue naıve population outside the ambit of its applicationdue to safety concerns and poor efficacy
Another phase 3 efficacy trial of CYD-TDV was car-ried out in five dengue endemic Latin American countriesHealthy children between the ages of 9 and 16 years wererandomly assigned in a 2 1 ratio to receive three doses ofthe vaccine or placebo at 0 6 and 12 months under blindedconditions The subjects were followed up for 25 monthsSerotype-specific vaccine efficacywas found to be 503 (95CI 291 to 652) 423 (95 CI 140 to 611) 74 (95 CI619 to 824) and 777 (95 CI 602 to 880) for DENV-1DENV-2 DENV-3 and DENV-4 respectively A statisticallysignificant efficacy against DENV-2 was a big boost Thoughthe overall efficacy of the vaccine in virologically confirmeddengue caseswas 608 (95CI 520 to 68) it was found to below in dengue naıve population 432 (95CI minus615 to 800)[90] Since this study enrolled children 9ndash16 years old (9ndash11and 12ndash16 yearsrsquo cohorts) the relative risk of hospitalizationwas observed to be fairly low (053) in year 3 of the follow-up But consistent with the Asian-Pacific trials the vaccineefficacy was found to be lower in dengue naıve vaccineesTheoverall efficacy was 837 (95 CI 622 to 937) and a low432 (95CIminus616 to 800) for dengue serotype positive andnaıve vaccinees respectively [89]
55 Licensed in Mexico Brazil and Philippines Dengvaxiahas received regulatory approvals in Mexico Brazil El Sal-vador and Philippines for administration in adults aged 9ndash45 years [4] because of the increased risk of hospitalizationobserved in children lt9 years old Moreover Dengvaxia wasfound to be poorly efficacious in naıve individuals whichrestricts its applicability to dengue endemic nations
56 Challenges and Obstacles in Developing Dengue Vac-cine The lower efficacy of Dengvaxia against dengue naıveindividuals has raised many issues on ADE Most of thecurrent vaccine candidates (eg LAV inactivated virus andchimeric viruses) carry all the cross-reactive epitopes leadingto generation of high quantity of cross-reactive antibodies(as compared to serotype-specific antibodies) Such an imbal-anced response overwhelmed with poorly neutralizing cross-reactive antibodies can cause ADE reducing the efficacyagainst the virus in vivo
Recent study using AG129 mouse lethal model showedthat inoculation with virus immune complexes (ICs) formedwith high quantity of highly neutralizing cross-reactive Abscaused lethal infection even though peak viremia level was
low On the other hand those formed with serotype-specificneutralizing antibodies (anti-domain III used in the study)did not cause any mortality at any concentration [20] Thisindicates that serotype specificity of antibodies elicited canbe crucial in deciding the efficacy of a vaccine candidateHowever recent data suggests that dengue vaccines are at acrossroad even with modest efficacy [89 91] NeverthelessWHO recommends development of an alternative denguevaccine candidate which is designed to elicit strongly neu-tralizing antibodies in absence of cross-reactive enhancingantibodies Such a vaccine candidate would enable higherefficacy and applicability to a broader group of subjectsincluding infants and naıve population
6 Conclusion
The absolute need for an efficacious tetravalent DENV vac-cine lack of an adequate animal disease model and immunecorrelates of diseases protection remain as some of the majorobstacles in developing a successful dengue vaccine Sincethe wild type mice do not replicate clinical signs of humandengue infection genetically engineered mouse models havebeen developed with considerable success to mimic someaspects of human infection The most successful system hasbeen the use of mouse-adapted DENV-2 and AG129 micethat lack IFN-120572120573120574 receptors Due to suppression of IFNpathway an important branch of host immune responseis disabled which allows DENV to replicate AG129 miceon infection with mouse-adapted DENV-2 develop vascularleakage without neurological complications thus mimickinghuman clinical signs of severe dengue Moreover this mousemodel has been found to be useful in scoring ADE bypassive transfer of anti-DENV antibodies and challenge withnonlethal dose of mouse-adapted DENV-2 The passivelytransferred antibodies are said to enhance the disease if themice succumb to infection and die Since mouse-adaptedDENVs are not the naturally circulating strains AG129 miceare being explored as a suitable dengue model with clinicalisolates too [92]With respect to evaluation of dengue vaccinecandidates also AG129mousemodel has been recommendedby WHO It should be noted that this model allows limitedevaluation since it lacks both type I and type II IFN pathwaysHence this limits production of high titer neutralizing anti-bodies which may further result in ADE [93]Thus extensivework is ongoing to further advance these mouse models toenable better extrapolation of mice data to humans
Sanofi Pasteur dengue vaccine Dengvaxia has now beenlicensed in a few countries but it recorded poor efficacy indengue naıve individuals during phase III evaluation Thiscould be due to a number of reasons It possesses yellowfever virus backbone and therefore lacks the critical dengueT cell epitopes of the nonstructural region which havebeen reported to play a vital role in providing protectionagainst dengue [94 95] Studies also implicate immunity todengue NS1 to be essential in providing protection [96 97]which it lacks The observation that it led to enhancement ofdisease [86] indicated that it generates a lot of cross-reactivenonneutralizingenhancing antibodies Thus not only thepresence of DENV neutralizing antibodies but also DENV
Journal of Immunology Research 11
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
[1] R Chen and N Vasilakis ldquoDengue-Quo Tu et Quo VadisrdquoViruses vol 3 no 9 pp 1562ndash1608 2011
[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
serotype-specific neutralizing antibodies may be the key to asuccessful dengue vaccine candidate Predominant immuneresponses to a natural DENV infection are highly cross-reactive in the presence of very limited serotype-specificneutralizing antibodiesThis could be considered as immuneevasion or disease enhancement strategy of DENVs Immuneresponses elicited by most dengue vaccine approaches basedon thewhole virusmay be similar to naturalDENV infectionsand thus disease or immune enhancement strategies (pre-dominant serotype cross-reactive neutralizing antibodies)of DENV may overcome the protective (minor serotype-specific neutralizing antibodies) efficacy of the whole virusbased vaccine candidate An effective dengue vaccinemust bedesigned which is capable of eliciting predominantly DENVserotype-specific neutralizing (protective) antibodies in theabsence of serotype cross-reactive neutralizing (disease-enhancing) antibodies The pipeline of dengue vaccinesis growing and notwithstanding lower efficacy a denguevaccine may soon become available for human use
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge support from Department of Bio-technology Council of Scientific and Industrial ResearchGovernment of India
References
[1] R Chen and N Vasilakis ldquoDengue-Quo Tu et Quo VadisrdquoViruses vol 3 no 9 pp 1562ndash1608 2011
[2] B R Murphy and S S Whitehead ldquoImmune response to den-gue virus and prospects for a vaccinerdquo Annual Review ofImmunology vol 29 pp 587ndash619 2011
[3] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006
[4] Sanofi Pasteur Media Release httpwwwsanofipasteurcomenarticlesFirst-Vaccinations-against-Dengue-Mark-Historic-Moment-in-Prevention-of-Infectious-Diseasesaspx
[5] World Health Organization Dengue Guidelines for DiagnosisTreatment Prevention andControlWHOGeneva Switzerland2009
[6] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013
[7] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012
[8] G N Malavige S Fernando D J Fernando and S L Senevi-ratne ldquoDengue viral infectionsrdquo Postgraduate Medical Journalvol 80 no 948 pp 588ndash601 2004
[9] J D Stanaway D S Shepard E A Undurraga et al ldquoTheglobal burden of dengue an analysis from the Global Burden
of Disease Study 2013rdquo The Lancet Infectious Diseases vol 16no 6 pp 712ndash723 2016
[10] G Rezza ldquoAedes albopictus and the reemergence of denguerdquoBMC Public Health vol 12 article 72 2012
[11] R Perera and R J Kuhn ldquoStructural proteomics of denguevirusrdquo Current Opinion in Microbiology vol 11 no 4 pp 369ndash377 2008
[12] S Apte-Sengupta D Sirohi and R J Kuhn ldquoCoupling of rep-lication and assembly in flavivirusesrdquo Current Opinion in Virol-ogy vol 9 pp 134ndash142 2014
[13] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002
[14] R Perera M Khaliq and R J Kuhn ldquoClosing the door onflaviviruses entry as a target for antiviral drug designrdquoAntiviralResearch vol 80 no 1 pp 11ndash22 2008
[15] Y Modis S Ogata D Clements and S C Harrison ldquoVariablesurface epitopes in the crystal structure of dengue virus type3 envelope glycoproteinrdquo Journal of Virology vol 79 no 2 pp1223ndash1231 2005
[16] R de Alwis M Beltramello W B Messer et al ldquoIn-depth anal-ysis of the antibody response of individuals exposed to primarydengue virus infectionrdquo PLoS Neglected Tropical Diseases vol 5no 6 Article ID e1188 2011
[17] W B Messer R de Alwis B L Yount et al ldquoDengue virusenvelope protein domain III hinge determines long-livedserotype-specific dengue immunityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no5 pp 1939ndash1944 2014
[18] R De Alwis S A Smith N P Olivarez et al ldquoIdentification ofhumanneutralizing antibodies that bind to complex epitopes ondengue virionsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 19 pp 7439ndash74442012
[19] G Fibriansah K D Ibarra T-S Ng et al ldquoCryo-EM structureof an antibody that neutralizes dengue virus type 2 by locking Eprotein dimersrdquo Science vol 349 no 6243 pp 88ndash91 2015
[20] SWatanabe KWKChan JWang L Rivino S-M Lok and SG Vasudevan ldquoDengue virus infection with highly neutralizinglevels of cross-reactive antibodies causes acute lethal smallintestinal pathology without a high level of viremia in micerdquoJournal of Virology vol 89 no 11 pp 5847ndash5861 2015
[21] W Dejnirattisai A Jumnainsong N Onsirisakul et al ldquoCross-reacting antibodies enhance dengue virus infection in humansrdquoScience vol 328 no 5979 pp 745ndash748 2010
[22] C De La Guardia and R Lleonart ldquoProgress in the identifica-tion of dengue virus entryfusion inhibitorsrdquo BioMed ResearchInternational vol 2014 Article ID 825039 13 pages 2014
[23] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004
[24] S-J LWu G Grouard-VogelW Sun et al ldquoHuman skin Lang-erhans cells are targets of dengue virus infectionrdquo Nature Med-icine vol 6 no 7 pp 816ndash820 2000
[25] A Molina-Cruz L Gupta J Richardson K Bennett W BlackIV and C Barillas-Mury ldquoEffect of mosquito midgut trypsinactivity on dengue-2 virus infection and dissemination inAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 72 no 5 pp 631ndash637 2005
12 Journal of Immunology Research
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
[26] M I Salazar J H Richardson I Sanchez-Vargas K E OlsonandB J Beaty ldquoDengue virus type 2 replication and tropisms inorally infected Aedes aegypti mosquitoesrdquo BMC Microbiologyvol 7 article 9 2007
[27] S Swaminathan and N Khanna ldquoExperimental dengue vac-cinesrdquo Molecular Vaccines From Prophylaxis to Therapy vol 1pp 135ndash151 2013
[28] J Barniol R Gaczkowski E V Barbato et al ldquoUsefulness andapplicability of the revised dengue case classification by diseasemulti-centre study in 18 countriesrdquoBMC InfectiousDiseases vol11 article 106 2011
[29] F Narvaez G Gutierrez M A Perez et al ldquoEvaluation of thetraditional and revised WHO classifications of dengue diseaseseverityrdquo PLoS Neglected Tropical Diseases vol 5 no 11 ArticleID e1397 2011
[30] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010
[31] S-WWan C-F Lin SWang et al ldquoCurrent progress in denguevaccinesrdquo Journal of Biomedical Science vol 20 no 1 article 372013
[32] N Bhamarapravati and Y Sutee ldquoLive attenuated tetravalentdengue vaccinerdquoVaccine vol 18 supplement 2 pp 44ndash47 2000
[33] N Bhamarapravati S Yoksan T Chayaniyayothin S Angsub-phakorn andA Bunyaratvej ldquoImmunizationwith a live attenu-ated dengue-2-virus candidate vaccine (16681-PDK 53) clinicalimmunological and biological responses in adult volunteersrdquoBulletin of theWorld Health Organization vol 65 no 2 pp 189ndash195 1987
[34] C Balas A Kennel F Deauvieau et al ldquoDifferent innate sig-natures induced in human monocyte-derived dendritic cells bywild-type dengue 3 virus attenuated but reactogenic dengue 3vaccine virus or attenuated nonreactogenic dengue 1ndash4 vaccinevirus strainsrdquo Journal of Infectious Diseases vol 203 no 1 pp103ndash108 2011
[35] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009
[36] VWatanaveeradej S Simasathien A Nisalak et al ldquoSafety andimmunogenicity of a tetravalent live-attenuated dengue vaccinein flavivirus-naive infantsrdquo The American Journal of TropicalMedicine and Hygiene vol 85 no 2 pp 341ndash351 2011
[37] S J Thomas K H Eckels I Carletti et al ldquoA phase IIrandomized safety and immunogenicity study of a re-derivedlive-attenuated dengue virus vaccine in healthy adultsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no1 pp 73ndash88 2013
[38] K Bauer I O Esquilin A S Cornier et al ldquoA phase IIrandomized safety and immunogenicity trial of a re-derivedlive-attenuated dengue virus vaccine in healthy children andadults living in puerto ricordquo The American Journal of TropicalMedicine and Hygiene vol 93 no 3 pp 441ndash453 2015
[39] S S Whitehead B Falgout K A Hanley J E Blaney Jr LMarkoff and B R Murphy ldquoA live attenuated dengue virustype 1 vaccine candidate with a 30-nucleotide deletion in the31015840 untranslated region is highly attenuated and immunogenicin monkeysrdquo Journal of Virology vol 77 no 2 pp 1653ndash16572003
[40] S S Whitehead ldquoDevelopment of TV003TV005 a singledose highly immunogenic live attenuated dengue vaccine what
makes this vaccine different from the Sanofi-Pasteur CYDtradevaccine rdquo Expert Review of Vaccines vol 15 no 4 2015
[41] B D Kirkpatrick S S Whitehead K K Pierce et al ldquoThe liveattenuated dengue vaccine TV003 elicits complete protectionagainst dengue in a human challenge modelrdquo Science Transla-tional Medicine vol 8 no 330 Article ID 330ra36 2016
[42] A R Precioso R Palacios B Thome G Mondini P Bragaand J Kalil ldquoClinical evaluation strategies for a live attenuatedtetravalent dengue vaccinerdquo Vaccine vol 33 no 50 pp 7121ndash7125 2015
[43] F Guirakhoo R Weltzin T J Chambers et al ldquoRecombinantchimeric yellow fever-dengue type 2 virus is immunogenic andprotective in nonhuman primatesrdquo Journal of Virology vol 74no 12 pp 5477ndash5485 2000
[44] B Guy M Saville and J Lang ldquoDevelopment of sanofi pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010
[45] C Y-H Huang S Butrapet K R Tsuchiya N BhamarapravatiD J Gubler and R M Kinney ldquoDengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine developmentrdquoJournal of Virology vol 77 no 21 pp 11436ndash11447 2003
[46] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo American Journal of Tropical Medicine and Hygienevol 84 no 6 pp 978ndash987 2011
[47] J E Osorio I D Velez C Thomson et al ldquoSafety andimmunogenicity of a recombinant live attenuated tetravalentdengue vaccine (DENVax) in flavivirus-naive healthy adults inColombia a randomised placebo-controlled phase 1 studyrdquoThe Lancet Infectious Diseases vol 14 no 9 pp 830ndash838 2014
[48] Takeda Efficacy Safety and Immunogenicity of TakedarsquosTetravalent Dengue Vaccine (TDV) in Healthy Children(TIDES) ClinicalTrialsgov Bethesda Md USA NationalLibrary of Medicine 2000 httpsclinicaltrialsgovct2showNCT02747927
[49] R Putnak D A Barvir J M Burrous et al ldquoDevelopmentof a purified inactivated dengue-2 virus vaccine prototype inVero cells immunogenicity and protection in mice and rhesusmonkeysrdquoThe Journal of Infectious Diseases vol 174 no 6 pp1176ndash1184 1996
[50] J R Putnak B-AColler GVoss et al ldquoAn evaluation of denguetype-2 inactivated recombinant subunit and live-attenuatedvaccine candidates in the rhesus macaque modelrdquo Vaccine vol23 no 35 pp 4442ndash4452 2005
[51] M Simmons T Burgess J Lynch and R Putnak ldquoProtectionagainst dengue virus by non-replicating and live attenuatedvaccines used together in a prime boost vaccination strategyrdquoVirology vol 396 no 2 pp 280ndash288 2010
[52] US Army Medical Research and Materiel Command ldquoSafetystudy of a vaccine (DENV-1 PIV) to prevent dengue dis-ease (DENV-1 PIV)rdquo in ClinicalTrialsgov National Library ofMedicine (US) Bethesda Md USA 2000 NLM IdentifierNCT01502735 httpsclinicaltrialsgovct2showNCT01502735
[53] US Army Medical Research and Materiel Command A Two-dose Primary Vaccination Study of a Tetravalent Dengue VirusPurified Inactivated Vaccine vs Placebo in Healthy Adults(DPIV-001) In ClinicalTrialsgov [Internet] Bethesda MdUSA National Library ofMedicine (US) 2000- [cited 2016May13] NLM Identifier NCT01666652 httpsclinicaltrialsgovct2showNCT01666652
[54] US Army Medical Research and Materiel Command ldquoATwo-dose Primary Vaccination Study of a Tetravalent Dengue
Journal of Immunology Research 13
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
Virus Purified Inactivated Vaccine vs Placebo in HealthyAdults (in Puerto Rico) (DPIV-002)rdquo ClinicalTrialsgovBethesda Md USA National Library of Medicine NLM Iden-tifier NCT01702857 2000 httpsclinicaltrialsgovct2showNCT01702857
[55] US Army Medical Research and Materiel Command TDENVPIV and LAV Dengue Prime-boost Strategy ClinicalTrialsgov Bethesda Md USA National Library of Medicine (US)NLM Identifier NCT02239614 2000 httpsclinicaltrialsgovct2showNCT02239614
[56] D E Clements B-A G Coller MM Lieberman et al ldquoDevel-opment of a recombinant tetravalent dengue virus vaccineimmunogenicity and efficacy studies in mice and monkeysrdquoVaccine vol 28 no 15 pp 2705ndash2715 2010
[57] D Govindarajan SMeschino L Guan et al ldquoPreclinical devel-opment of a dengue tetravalent recombinant subunit vaccineimmunogenicity and protective efficacy in nonhuman pri-matesrdquo Vaccine vol 33 no 33 pp 4105ndash4116 2015
[58] S B Manoff S L George A J Bett et al ldquoPreclinical andclinical development of a dengue recombinant subunit vaccinerdquoVaccine vol 33 no 50 pp 7126ndash7134 2015
[59] Merck SharpampDohmeCorp Study of aDengueVaccine (V180)in Healthy Adults (V180-001) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01477580 2000 httpsclinicaltrialsgovct2showNCT01477580
[60] M Simmons G S Murphy and C G Hayes ldquoShort reportantibody responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccinerdquo The AmericanJournal of Tropical Medicine and Hygiene vol 65 no 2 pp 159ndash161 2001
[61] L Hermida R Rodrıguez L Lazo et al ldquoA fragment of theenvelope protein from dengue-1 virus fused in two differentsites of themeningococcal P64k protein carrier induces a func-tional immune response in micerdquo Biotechnology and AppliedBiochemistry vol 39 no 1 pp 107ndash114 2004
[62] L Hermida R Rodrıguez L Lazo et al ldquoA dengue-2 Envelopefragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune responseagainst the virus in micerdquo Journal of Virological Methods vol115 no 1 pp 41ndash49 2004
[63] L Hermida L J Bernardo M Martın et al ldquoA recombinantfusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhumanprimatesrdquo Vaccine vol 24 no 16 pp 3165ndash3171 2006
[64] J R Danko C G Beckett and K R Porter ldquoDevelopment ofdengue DNA vaccinesrdquo Vaccine vol 29 no 42 pp 7261ndash72662011
[65] C G Beckett J Tjaden T Burgess et al ldquoEvaluation of aprototype dengue-1 DNA vaccine in a Phase 1 clinical trialrdquoVaccine vol 29 no 5 pp 960ndash968 2011
[66] K R Porter D Ewing L Chen et al ldquoImmunogenicity andprotective efficacy of a vaxfectin-adjuvanted tetravalent dengueDNA vaccinerdquo Vaccine vol 30 no 2 pp 336ndash341 2012
[67] K Raviprakash T Luke J Doukas et al ldquoA dengue DNAvaccine formulated with Vaxfectinreg is well tolerated and elicitsstrong neutralizing antibody responses to all four dengueserotypes in New Zealand white rabbitsrdquo Human Vaccines andImmunotherapeutics vol 8 no 12 pp 1764ndash1768 2012
[68] US Army Medical Research and Materiel Command Evalua-tion of the Safety and the Ability of a DNA Vaccine to Protect
Against Dengue Disease (TVDV) ClinicalTrialsgov BethesdaMd USA National Library of Medicine (US) NLM Iden-tifier NCT01502358 2000 httpsclinicaltrialsgovct2showNCT01502358
[69] K Raviprakash D Wang D Ewing et al ldquoA tetravalent denguevaccine based on a complex adenovirus vector provides signifi-cant protection in rhesus monkeys against all four serotypes ofdengue virusrdquo Journal of Virology vol 82 no 14 pp 6927ndash69342008
[70] L J White M M Parsons A C Whitmore B M Williams Ade Silva and R E Johnston ldquoAn immunogenic and protectivealphavirus replicon particle-based dengue vaccine overcomesmaternal antibody interference in weanling micerdquo Journal ofVirology vol 81 no 19 pp 10329ndash10339 2007
[71] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007
[72] D H Holman D Wang K Raviprakash et al ldquoTwo com-plex adenovirus-based vaccines that together induce immuneresponses to all four dengue virus serotypesrdquo Clinical andVaccine Immunology vol 14 no 2 pp 182ndash189 2007
[73] N U Raja D H Holman DWang et al ldquoInduction of bivalentimmune responses by expression of dengue virus type 1 and type2 antigens from a single complex adenoviral vectorrdquo AmericanJournal of Tropical Medicine andHygiene vol 76 no 4 pp 743ndash751 2007
[74] R Suzuki E R Winkelmann and P W Mason ldquoConstructionand characterization of a single-cycle chimeric flavivirus vac-cine candidate that protects mice against lethal challenge withdengue virus type 2rdquo Journal of Virology vol 83 no 4 pp 1870ndash1880 2009
[75] S Mani L Tripathi R Raut et al ldquoPichia pastoris-expresseddengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibod-iesrdquo PLoS ONE vol 8 no 5 Article ID e64595 2013
[76] T J Chambers A Nestorowicz P W Mason and C M RiceldquoYellow feverJapanese encephalitis chimeric viruses construc-tion and biological propertiesrdquo Journal of Virology vol 73 no4 pp 3095ndash3101 1999
[77] F Guirakhoo K Pugachev Z Zhang et al ldquoSafety and efficacyof chimeric yellow fever-dengue virus tetravalent vaccine for-mulations in nonhuman primatesrdquo Journal of Virology vol 78no 9 pp 4761ndash4775 2004
[78] B Guy V Barban N Mantel et al ldquoEvaluation of interferencesbetween dengue vaccine serotypes in a monkey modelrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no2 pp 302ndash311 2009
[79] S B Halstead ldquoIdentifying protective dengue vaccines guideto mastering an empirical processrdquo Vaccine vol 31 no 41 pp4501ndash4507 2013
[80] F Guirakhoo S Kitchener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicity effectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006
[81] D Morrison T J Legg C W Billings R Forrat S Yoksan andJ Lang ldquoA novel tetravalent dengue vaccine is well tolerated andimmunogenic against all 4 serotypes in flavivirus-naive adultsrdquoThe Journal of Infectious Diseases vol 201 no 3 pp 370ndash3772010
14 Journal of Immunology Research
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
[82] R Z Capeding I A Luna E Bomasang et al ldquoLive-attenuatedtetravalent dengue vaccine in children adolescents and adultsin a dengue endemic country randomized controlled phase Itrial in the Philippinesrdquo Vaccine vol 29 no 22 pp 3863ndash38722011
[83] G H Dayan M Thakur M Boaz and C Johnson ldquoSafety andimmunogenicity of three tetravalent dengue vaccine formula-tions in healthy adults in the USArdquo Vaccine vol 31 no 44 pp5047ndash5054 2013
[84] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012
[85] A Ghosh and L Dar ldquoDengue vaccines challenges develop-ment current status and prospectsrdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 3ndash15 2015
[86] S B Halstead and P K Russell ldquoProtective and immunologicalbehavior of chimeric yellow fever dengue vaccinerdquo Vaccine vol34 no 14 pp 1643ndash1647 2016
[87] L A Villar D M Rivera-Medina J L Arredondo-Garcıa etal ldquoSafety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds a randomized controlledphase II trial in Latin Americardquo Pediatric Infectious DiseaseJournal vol 32 no 10 pp 1102ndash1109 2013
[88] M R Capeding N H Tran S R S Hadinegoro et al ldquoClinicalefficacy and safety of a novel tetravalent dengue vaccine inhealthy children in Asia a phase 3 randomised observer-masked placebo-controlled trialrdquoTheLancet vol 384 no 9951pp 1358ndash1365 2014
[89] S R Hadinegoro J L Arredondo-Garcıa M R Capeding et alldquoEfficacy and long-term safety of a dengue vaccine in regions ofendemic diseaserdquoTheNewEngland Journal ofMedicine vol 373no 13 pp 1195ndash1206 2015
[90] L Villar G H Dayan J L Arredondo-Garcıa et al ldquoEfficacy ofa tetravalent dengue vaccine in children in Latin AmericardquoTheNew England Journal of Medicine vol 372 no 2 pp 113ndash1232015
[91] A Wilder-Smith and D J Gubler ldquoDengue vaccines at a cross-road despite modest ef cacy a newly developed vaccine may bekey for controlling denguerdquo Science vol 350 no 6261 pp 626ndash627 2015
[92] K W K Chan S Watanabe R Kavishna S Alonso and S GVasudevan ldquoAnimal models for studying dengue pathogenesisand therapyrdquo Antiviral Research vol 123 pp 5ndash14 2015
[93] R M Zellweger and S Shresta ldquoMouse models to study denguevirus immunology and pathogenesisrdquo Frontiers in Immunologyvol 5 article 151 2014
[94] D Weiskopf M A Angelo E L De Azeredo et al ldquoCompre-hensive analysis of dengue virus-specific responses supports anHLA-linked protective role for CD8+ T cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 22 pp E2046ndashE2053 2013
[95] D Weiskopf and A Sette ldquoT-cell immunity to infection withdengue virus in humansrdquo Frontiers in Immunology vol 5 article93 2014
[96] N Modhiran D Watterson D A Muller et al ldquoDengue virusNS1 protein activates cells via Toll-like receptor 4 and dis-rupts endothelial cellmonolayer integrityrdquo Science TranslationalMedicine vol 7 no 304 Article ID 304ra142 2015
[97] R P Beatty H Puerta-Guardo S S Killingbeck DGlasner andE Harris ldquoDengue virus NS1 triggers endothelial permeabilityand vascular leak that is prevented by NS1 vaccinationrdquo ScienceTranslationalMedicine vol 7 no 304 Article ID 304ra141 2015
![Dengue fever: Causes, complications, and vaccine strategies ......dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) which may turn fatal [2].](https://static.documents.pub/doc/80x56/5f57aec821c68c72c60d0d52/dengue-fever-causes-complications-and-vaccine-strategies-dengue-fever.jpg)
![Dengue Fever/Severe Dengue Fever/Chikungunya Fever · Dengue fever and severe dengue (dengue hemorrhagic fever [DHF] and dengue shock syndrome [DSS]) are caused by any of four closely](https://static.documents.pub/doc/80x56/5e87bf3e7a86e85d3b149cd7/dengue-feversevere-dengue-feverchikungunya-dengue-fever-and-severe-dengue-dengue.jpg)
