|Year : 2015 | Volume
| Issue : 1 | Page : 3-15
Dengue vaccines: Challenges, development, current status and prospects
A Ghosh, L Dar
Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
|Date of Submission||20-Jul-2014|
|Date of Acceptance||17-Sep-2014|
|Date of Web Publication||5-Jan-2015|
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
Infection with dengue virus (DENV) is the most rapidly spreading mosquito-borne viral disease in the world. The clinical spectrum of dengue, caused by any of the four serotypes of DENV, ranges from mild self-limiting dengue fever to severe dengue, in the form dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased rates of hospitalization due to severe dengue, during outbreaks, result in massive economic losses and strained health services. In the absence of specific antiviral therapy, control of transmission of DENV by vector management is the sole method available for decreasing dengue-associated morbidity. Since vector control strategies alone have not been able to satisfactorily achieve reduction in viral transmission, the implementation of a safe, efficacious and cost-effective dengue vaccine as a supplementary measure is a high public health priority. However, the unique and complex immunopathology of dengue has complicated vaccine development. Dengue vaccines have also been challenged by critical issues like lack of animal models for the disease and absence of suitable markers of protective immunity. Although no licensed dengue vaccine is yet available, several vaccine candidates are under phases of development, including live attenuated virus vaccines, live chimeric virus vaccines, inactivated virus vaccines, subunit vaccines, DNA vaccines and viral-vectored vaccines. Although some vaccine candidates have progressed from animal trials to phase II and III in humans, a number of issues regarding implementation of dengue vaccine in countries like India still need to be addressed. Despite the current limitations, collaborative effects of regulatory bodies like World Health Organization with vaccine manufacturers and policy makers, to facilitate vaccine development and standardize field trials can make a safe and efficacious dengue vaccine a reality in near future.
Keywords: Challenges, dengue vaccine, prospect India, status
|How to cite this article:|
Ghosh A, Dar L. Dengue vaccines: Challenges, development, current status and prospects. Indian J Med Microbiol 2015;33:3-15
|How to cite this URL:|
Ghosh A, Dar L. Dengue vaccines: Challenges, development, current status and prospects. Indian J Med Microbiol [serial online] 2015 [cited 2020 Jun 4];33:3-15. Available from: http://www.ijmm.org/text.asp?2015/33/1/3/148369
| ~ Introduction|| |
Dengue, a mosquito-borne flaviviral infection caused by dengue virus (DENV) with four antigenically distinct serotypes (DENV1-4), has emerged as one of the most important vector-borne diseases of public health concern, particularly in the tropical and subtropical regions of the world. With almost 40% of the world's population residing in countries endemic for dengue, there has been a steady increase in global incidence of this infection. World Health Organization (WHO) estimates that 50-100 million dengue infections occur worldwide annually. , Explosive dengue epidemics are being reported every year from more than 100 endemic countries spanning South-East Asia, Western Pacific, Africa, the Americas and the East Mediterranean.  The disease has also spread to newer geographical locales mostly due to rise in air travel and international trade, particularly of used tyres. Local transmission of dengue was reported for the first time from European countries like France and Croatia in 2010 and imported cases have been detected in more than 10 countries in Europe in 2012. In 2013, cases of dengue have occurred in Florida, USA and Yunnan province of China, which were previously dengue free. ,
The clinical spectrum of dengue infection ranges from asymptomatic infection to uncomplicated classical dengue fever to severe dengue in the form dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Sequential infection with DENV of a different serotype leads to severe dengue characterised by capillary leakage, increase in haematocrit, thrombocytopenia, bleeding manifestations and progression to shock, requiring hospitalisation. There is no specific antiviral therapy and treatment is only supportive.  An estimated 5,00,000 people require hospital admission each year due to DHF/DSS, a major proportion of whom are children. Although efficient case management and improvement in health-care facilities have brought the dengue case fatality rates to below 1% in various parts of the endemic areas, case fatality rates during focal outbreaks away from urban areas can be as high as 5%.  With sharp rise in rates of hospital admission due to DHF/DSS, dengue also poses a heavy economic burden to health system and society. In a prospective study, involving eight countries in Asia and Latin America, which has looked into the economic costs of dengue, the mean cost per hospitalised case of dengue well exceeded 500 US dollars.  In addition dengue also accounts for an overwhelmingly high annual global burden of 7, 50, 000 disability adjusted life years. ,
To mitigate the ever-increasing social and economic burden of dengue, WHO targets to reduce the overall dengue mortality and morbidity by 50% and 25%, respectively, by 2020.  In the absence of specific antiviral therapy, effective vector control is the only strategy available to achieve and sustain reduction of mortality and morbidity rates due to dengue. Vector control interventions have not been satisfactorily effective in reducing dengue transmission due to increasing rural-urban migration, rapid population growth, unplanned urbanisation and the emergence of insecticide resistance in mosquitoes. , Hence, a safe, efficacious and a cost-effective dengue vaccine can be a supplementary measure in dengue prevention and control strategy in endemic areas. In addition, vaccination of target groups like travellers to endemic areas and migratory population can be a suitable strategy in preventing the spread of dengue to non-endemic or dengue-free regions.
Dengue vaccine development is receiving increasing interest and support by researchers, vaccine manufacturers, policy makers and funding agencies. In this review, we discuss the challenges for developing an ideal dengue vaccine and the current status of dengue vaccine candidates in different phases of development. The review also highlights the prospect of a dengue vaccine in India in terms of its application as a part of preventive strategy against dengue.
| ~ Challenges in Development of an Ideal Dengue Vaccine|| |
An ideal dengue vaccine should produce life-long protective immune response in the form of neutralising antibodies that are equally effective against all the four serotypes of dengue virus (DENV 1 to 4). The issue of eliciting equal protection against all dengue virus serotypes is crucial because of the phenomenon of antibody-dependent enhancement (ADE). , Immune enhancement or ADE is observed in dengue virus infection due to heterologous non-neutralising antibodies and carries the potential risk of precipitating serious manifestations like dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS) during secondary infection by a different serotype. A primary infection with one serotype of the virus produces long-term protective immunity to that homologous serotype. This is mediated by serotype-specific neutralising antibodies that block viral attachment, prevent fusion of the virus membrane with endocytic vacuole membrane, inhibit release of viral RNA into the cytoplasm and promote complement-mediated lysis of antibody-coated virus. , In contrast, heterologous immunity to the serotypes other than the infecting serotype wanes over a short period of time. Pre-existing non-neutralising or sub-neutralising heterotypic antibodies from a previous DENV infection allows the virus during a subsequent heterotypic infection, to bind to surface expressed Fc gamma (Fcg) receptors on monocytes and macrophages. Enhanced Fcg-receptor binding enables the virus to gain access to these cells, which finally results in increased viral replication and a high level of viremia of the heterotypic virus, a feature that has been strongly associated with severe disease (DHF/DSS). ,, Since multiple serotypes of DENV may co-circulate in endemic areas, vaccination strategies should aim to induce a balanced long-lasting protective immune response to each of the four serotypes, which justifies the usage of tetravalent formulations.  Sequential, rather than simultaneous induction of neutralising antibody response to each of the four DENV serotypes may expose the vaccine recipients to an increased risk of ADE for a certain period of time upon challenge with a wild DENV to which the antibody levels are still sub-neutralising.
A hypothetical risk of ADE is associated with live attenuated dengue vaccines during the brief period of viremia post-immunisation. In dengue endemic areas, already circulating heterotypic antibodies in vaccinees in sub-neutralising concentrations can augment the level of replication of the vaccine virus strains. However, the risk of vaccine virus causing severe disease, due to high levels of multiplication, is very low due to the attenuated phenotype of the vaccine strains.  However, a major safety issue due to ADE has been observed in vaccination with multivalent live attenuated DENV vaccines due to the phenomenon of "viral interference". , Interference has been attributed to increased replication of one serotype of live attenuated DENV strain in the multivalent formulation that leads to unequal levels of neutralising antibodies against the 4 DENV serotypes. Prevention of interference, by empirical adjustment of the doses of each of the four DENV serotypes still remains a challenge in development of a tetravalent live attenuated DENV vaccine. ,, In addition, antibody responses waning below protective levels over time also increases the possibilities of immune enhancement following natural infection with wild DENV.  This warrants the usage of appropriately timed multiple doses of dengue vaccine. Since majority of areas endemic for dengue are located in developing countries, the issue of cost-effective implementation and uninterrupted availability of dengue vaccine in multiple doses remains an unanswered challenge in these resource-limited health-care settings. ,
One of the major challenges that have hindered the development ofa safe and effective dengue vaccine is the lack of an appropriate animal model that elucidates the pathogenesis, immune response and clinical course of dengue infection in humans. Methods like inoculation of mouse-adapted DENV strain into suckling micevia intracerebral route leading to paralysis and death of the animal or into immune-deficient animals like AG129 mice lacking interferon-receptor genes or severe combined immunodeficient (SCID) mice engrafted with human carcinoma or dendritic cells, are unsuitable for studying immune responses in dengue vaccine pre-clinical trials. ,, Non-human primates (NHPs), who show dengue viremia kinetics closely similar to that in humans, have been widely used for pre-clinical evaluation of dengue vaccines. ,, However, the utility of NHP models is only limited to the assessment of reduction in viremia since clinical features of dengue ranging from uncomplicated fever to DHF and DSS are not manifested in NHPs. Thus the risk of developing severe dengue due to ADE during secondary infection after vaccination cannot be evaluated using NHP models. 
Absence of established immune correlates of protection is yet another limitation. Neutralising antibody response to a specific DENV serotype is traditionally detected by the plaque reduction neutralisation test (PRNT), presented as a reciprocal of antibody dilution at which 50-90% (PRNT 50 or PRNT 90 ) of viral infectivity is neutralised. A cut-off of PRNT titre of ≥10 is taken as the marker for the presence of neutralising antibodies to a given serotype.  However, NHPs with PRNT titre for DENV-3 apparently adequate for protective immune response, post-immunisation, suffered from breakthrough viremia upon challenge with the same DENV serotype.  The PRNTs used for evaluation of dengue vaccines in clinical trials were also unable to distinguish homotypic from heterotypic immunity. , One of the drawbacks of PRNT is that the assay is performed on cell lines like Vero or LLC-MK 2 which lack surface expressed Fcg receptors. Since cells of monocyte and macrophage lineages, the target cells for DENV replication in human body, abundantly express Fcg receptors, which allow DENV entry into these cells, immunodiagnostic assays performed on cells lacking Fcg receptors may not be true predictors of protective immune response. ,
An additional problem with PRNT is the occurrence of interlaboratory variations, which may account for erroneous results in vaccine efficacy trials.  Standardised guidelines set up by regulatory bodies like WHO for performing PRNT thus, need to be strictly followed. In addition, no standardised immunoassays are available till date, which can reliably measure the T-cell-mediated immune response to non-structural proteins during a heterologous DENV infection. Measurement of T-cell response is important because of the phenomenon of original antigenic sin in which memory CD8+ T-cell response to the non-structural antigen-3 (NS-3) of a DENV during secondary infection with a different serotypeleads to a deleterious tumour necrosis factor-α (TNF-α) predominant inflammatory response, a key feature of DHF and DSS. ,
An important determinant of the outcome of vaccine efficacy during dengue vaccine clinical trials is the selection of primary efficacy end-point. Primary efficacy end-point for a dengue vaccine trial can be either confirmation of the presence of DENV in all patients with febrile illness in the test population or only in patients with features of severe dengue. Although detecting DENV in severe dengue suspects based on hospital-based surveillance is apparently an easy cost-effective alternative for primary end-point, this method has some potential drawbacks. Selecting only severe dengue cases underestimates the overall burden of dengue in a given population. Moreover, because trial participants will be under surveillance and are likely to be treated early, the number of severe cases will tend to be lower, thus extending the trial for a longer duration. In a given population, laboratory confirmation of DENV infection by virus isolation, reverse transcriptase - polymerase chain reaction (RT-PCR) or antigen detection in all vaccines with febrile illness may be a better predictor of vaccine efficacy during clinical trial.  Detecting even the mild cases of dengue in a vaccinated population, by community-based active surveillance, can be a major challenge, in areas with limited health-care resources.
Despite the existing challenges for an ideal dengue vaccine, development of dengue vaccine candidates through various approaches have progressed over the last decade and field trials of these vaccine candidates are being conducted in both endemic and non-endemic areas. A classification of the current approaches for dengue vaccine development is shown in [Figure 1].
Live attenuated dengue virus vaccines
Live attenuated vaccine viruses are capable of producing robust immune responses and immune memory closely similar to that induced in natural infection by wild-type viruses. As discussed earlier, live attenuated dengue virus vaccines need to be tetravalent formulations producing a balanced neutralising immune response protective against all four serotypes. The development of live dengue virus vaccines is also guided by the following principles. For a live, attenuated dengue virus vaccine to be safe and non-reactogenic, the vaccine strains need to show restricted replication in humans to preclude the development of clinically symptomatic dengue infection in vaccinated individuals. However, subclinical infection characterised by mild rash, transient leucopenia and mild elevation of liver enzymes can occur even in presence of low levels of viral replication and are included as acceptable adverse reaction. , An acceptable level of viremia ranges from 10 0.5 to 10 2.5 plaque forming units (PFU)/ml as occurs for the live attenuated yellow fever vaccine.  Low levels of viremia in vaccines also ensure reduced transmission of vaccine viruses from humans to mosquitoes. In addition, it is necessary for the live attenuated vaccine viruses to show limited replicative power in mosquitoes, which also decreases the chances of vector borne transmission. The vaccine viruses should have high infectivity for humans in low doses so as to mount an adequate immune response and efficient replicative ability in cell culture that will allow cost-effective delivery of the vaccine. , For an ideal live, attenuated dengue vaccine it is crucial the mutations that result in viral attenuation remains genetically stable during replication in humans. Hence, it is required the genetic basis of attenuation be clearly defined for all the vaccine strains and monitored from the time of manufacture to its usage in humans. ,,
The earliest efforts of using live attenuated viruses in dengue vaccine formulations have been credited to Albert Sabin who attenuated the Hawaii strain of DENV by serial passage in suckling mouse brain as early as 1940s during World War II. This approach of preparing live attenuated vaccine from mouse brain and spinal cord extract was later abandoned, primarily because of safety concerns associated with the inoculation of mouse neural tissue.  Current methods of producing live attenuated viruses for dengue vaccines include attenuation by serial passage in cell lines, targeted mutagenesis and by constructing chimeric vaccine viruses.
Cell culture passage based live attenuated vaccine
Based on preliminary observation that low passage DENV isolates produced smaller plaques in cell lines, became temperature sensitive (inhibition of plaque formation at temperatures above 37°C) and showed less neurovirulence in suckling mice, development of live attenuated dengue vaccines by serial passage in cell lines was initiated at Mahidol University, Bangkok, Thailand. DENV-1, DENV-2 and DENV-4 strains were attenuated by 15 passages in primary dog kidney (PDK) cells, while DENV-3 which failed to replicate in PDK cells, was attenuated by 48 passages in primary African green monkey kidney cells and three final passages in foetal Rhesus monkey lung cells. ,, When the attenuated strain of each serotype was combined to form a tetravalent formulation, the vaccine failed to elicit a balanced immune response to all the serotypes despite modulating the viral concentrations. , Increased frequency of adverse reactions like fever, rash, myalgia and retro-orbital pain, primarily related to the DENV-3 vaccine strain prevented the Mahidol University live attenuated vaccine from further development and testing. ,
Efforts to prepare a live attenuated dengue vaccine based on cell culture passage based attenuated DENV strains were also undertaken at the Walter Reed Army Institute of Research (WRAIR), Maryland, USA. Candidate strains belonging to all DENV serotypes were attenuated by initial passages in PDK cells followed by final passages in foetal Rhesus monkey lung cells. Monovalent vaccine candidates were tested in NHP models and in a phase I clinical trial to select out the suitable strains and standardise the dosage. The passage level required for an optimal balance between immunogenicity and reactogenicity was also determined. , Based on the safety and immunogenicity data generated, four vaccine candidates, namely, DENV-1 45AZ5 PDK20, DENV-2 S16803 PDK50, DENV-3 CH53489 PDK20 and DENV-4 341750 PDK 20 (strain nomenclature: DENV serotype, strain number, passage number in PDK cell line) were selected for use in tetravalent formulations.  Sixteen tetravalent formulations based on combinations of high (10 5-6 pfu/dose) or low (10 3.5-4.5 pfu/dose) concentrations of each DENV serotype were prepared for inoculation in non-immune adult volunteers as a part of a phase I trial. Percentages of vaccinees showing neutralising antibody responses to each of DENV-1, DENV-2 and DENV-3 ranged from approximately 70%-80%. However, the rate of seroconversion for DENV-4 was significantly low (below 40%), which was attributed to over attenuation of the DENV-4 strain. Although all formulations were relatively safe, DENV-1 component was under attenuated, leading to increased reactogenicity of the vaccine.  Hence, to overcome the problem, a seventeenth formulation was developed which used a higher passage for DENV-1 and a lower passage for DENV-4. This formulation showed promising results, with 63% of flavivirus naïve adults in Puerto Rico developing neutralising antibody response after 2 doses of the vaccine given 6 months apart.  The vaccine was also well tolerated in a group of flavivirus naïve school children and toddlers. , WRAIR entered into a collaboration with GlaxoSmithKline (GSK) and started a phase II randomised observer-blind, placebo-controlled trial (Trial No: NCT00350337) in 2007 with two doses of the vaccine administered 6 months apart to healthy adults. Different formulations of the WRAIR-GSK live attenuated tetravalent DENV vaccine had shown to have acceptable safety and immunogenicity in small number of healthy subjects after two doses in phase II trial with tetravalent antibody rates of more than 60%.  However, results of evaluation in larger number of healthy adults and children are not available.
Targeted mutagenesis based live attenuated vaccine
Suboptimal immunogenicity due to accumulation of unpredictable mutations during cell culture passage, is a major disadvantage of tissue culture-based live attenuated vaccine.  To overcome this problem, a novel strategy was devised by the Laboratory of Infectious Disease at the National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), Maryland, USA, where attenuation of DENV strains were achieved by site directed mutagenesis. Targeted deletion of 30 nucleotides in the 3'- untranslated region (UTR) of full length complimentary DNA (cDNA) clones of DENV-1 and DENV-4 produced attenuated strains designated as DEN1Δ30 and DEN4Δ30, respectively. , Both strains, at a dose of 10 3 pfu, generated optimal neutralising antibody response in vaccinees with the side effects being a faint rash, transient leucopenia and mild increase in liver enzymes. The Δ30 mutant strains showed genetic stability and produced low levels of viremia in humans. Mosquito-borne transmission was absent for both the vaccine strains. , A major disadvantage was the lack of attenuation of DENV-2 and DENV-3 strains by the Δ30 mutation. Therefore, an alternative approach of attenuation for DENV-2 and DENV-3 was devised by constructing chimeric vaccine on a DEN4Δ30 backbone, which will be discussed later. 
Chimeric dengue vaccines
Production of chimeric vaccines is based on the substitution of specific protein encoding genes of one virus for those of another thereby, constructing a chimera virus. Dengue chimera vaccines can be classified into two groups (i) chimera of a wild type DENV with another attenuated flavivirus (ii) chimera of a wild type DENV with an attenuated DENV (intertypic chimera).
Chimera of a wild type DENV with another attenuated flavivirus strain: The method of generating recombinant live chimera monovalent DENV vaccine candidate strain is accomplished by replacement of pre-membrane (prM) and envelope (E) genes of a well-established attenuated flavivirus strain e.g. Yellow fever live attenuated vaccine virus strain 17D (YFD 17D), with the corresponding gene of wild type DENV.  The concept was based on the fact that protective immunity in dengue infection is mediated by neutralising antibodies against prM and E protein and chimeric viruses carrying the prM and E genes of wild DENV within the backbone of YFD17D could thus evoke a protective immune response in vaccinees. This novel technology, which originated at the US National Institutes of Health and St. Louis University Health Science Centre, was further developed at Acambis Inc., Paris, France (now acquired by Sanofi Pasteur, Pennsylvania, USA), where all four chimeric live attenuated dengue vaccine strains designated as CYD 1-4 have been combined into a tetravalent formulation. ,
All four dengue chimera vaccine candidates (CYD 1-4) showed high degree of genetic stability during passage in vero cells due to the high fidelity of RNA polymerase of the YFD 17D strain. , In addition, plaque morphology monitoring of CYD 1-4 demonstrated in vitro phenotypic stability of the vaccine viruses as well.  CYD 1-4 were able to infect dendritic cells in vitro, just like the wild-type DENV in course of natural infection. Dendritic cell maturation by the chimeric viruses induced type I interferon response, with limited production of inflammatory cytokines, thereby proving the good safety profile of these vaccine candidates.  Lack of cross-reactivity of non-structural 3 (NS-3) antigen of DENV with that of the YFD 17D strain eliminated the possibility of a heterologous anti-NS3 CD8+ T-cell-mediated deleterious inflammatory cytokine response during dengue infection following vaccination with a chimera virus expressing NS-3 of YFD17D strain. ,
Pre-clinical evaluation of different tetravalent formulations of the chimera viruses in NHPs, like cynomolgus monkeys, demonstrated high rates of seroconversion and post-vaccination protection against all DENV serotypes. Viral interference was observed in Rhesus macaques post-vaccination, which was bypassed using bivalent formulations in two separate sites in multiple doses. , The safety and immunogenicity of CYD were established by testing monovalent chimeras in phase I trial in humans. Pre-existing immunity to yellow fever virus did not interfere with dengue seroconversion. Instead it resulted in production of long lasting cross-neutralising antibody response to all 4 DENV serotypes.  Subsequently, tetravalent formulations containing 5log 10 cell culture infective dose 50 (CCID 50 ) were administered in dengue naïve US adults. Seroconversion was observed to 4 DENV serotypes in all vaccinees after three doses, with the first dose being associated with low levels of viremia and a serotype-4 predominant antibody response. An initial DENV-4 antibody response did not cause ADE-mediated viremia of other serotypes after subsequent doses.  In dengue-endemic areas like Philippines, the phase I trial included both children and adults. Rates of seropositivity against all DENV serotypes ranged from 91 to 100% in 2-5-years old to 88-96% in 6-11-year old and adolescents and 100% in adults.  The safety profile of the tetravalent vaccine was equally good in both endemic and non-endemic areas. ,
Various tetravalent formulations of CYD viruses in different doses were tested in a randomised, double-blind multicenter phase IIb trial in healthy US adults. Although the safety and reactogenicity profiles were similar for all vaccine formulations, the tetravalent vaccine containing 5log 10 tissue culture infective dose 50 (TCID 50 ) of each serotype (CYD-TDV5555) demonstrated the best immunogenicity among all, which strongly favoured further development of this formulation.  In another randomised, controlled phase II trial (registered at ClinicalTrials.gov, NCT00842530) in 4-11-year-old school children in Ratchaburi province, Thailand, the efficacy of CYD-TDV after three doses was found to be 30.2% and variable across different serotypes. The study revealeda vaccine efficacy of approximately 61%, 82% and 90% against DENV-1, DENV-3 and DENV-4, respectively, and only 3.5% against DENV-2 after at least a single dose. Multiple doses failed to improve the protective efficacy, with just 9.2% efficacy against DENV-2 after three doses.  The lack of efficacy against DENV-2 was attributed to two reasons. First, the Asian 1 genotype of DENV-2 circulating in Thailand had mutations in E protein that resulted in an antigenic mismatch with the CYD-2 vaccine virus, in which the donor wild type DENV-2 belonged to Asian/American lineage. , Secondly, neutralising antibody response in the trial was determined by PRNT in Vero cells, which are Fcϒ-receptor negative. As discussed earlier, neutralising antibodies detected by PRNT in Fcϒ-receptor negative cells may not truly predict vaccine efficacy since the ADE-mediated increase in viral replication in human body occurs in Fcϒ-receptor bearing monocyte and macrophages. 
A randomised, blinded, controlled phase II trial was also conducted upon 9-16-year-old subjects from Latin American countries like Columbia, Honduras, Mexico and Puerto Rico (registered at Clinical Trials.gov, NCT00993447). After three doses of CYD-TDV, the seropositivity for at least two, three or all four serotypes were 100%, 90.6% and 93.4% respectively. Antibody titres were higher in vaccinees, who were seropositive for flavivirus antibodies in the baseline, compared to flavivirus seronegative subjects. The rates of virologically confirmed dengue cases for all DENV serotypes, including DENV-2, was lower in vaccine group compared to that in control group.  The results of this trial were different from that of the phase II trial conducted in Thailand. The contrast in results was attributed to the difference in epidemiology and circulating virus strain differences between Asian and Latin American countries.  Further studies in different epidemiological settings involving larger study population and better methods for measuring protective immune response can answer the queries raised regarding the efficacy of this vaccine. However, CYD-TDV (Chimeravax-Den) is undergoing phase III clinical trial in Asia, Australia and Latin America (registered at ClinicalTrials.gov, NCT01373281, NCT01134263and NCT01374516, respectively), which may provide crucial data about the protective efficacy of this vaccine. 
Dengue virus intertypic chimera: Production of intertypic chimeras is based on the introduction of prM and E genes of wild-type DENV strains into the cDNA derived from a well-characterised DENV strain. One of the attenuated DENV strains that has been used for the production of intertypic chimeras is the DENV-2 PDK-53 derived at Mahidol University, Bangkok, Thailand by 53 serial passages in primary dog kidney cells of a wild-type DENV-2 strain. Inoculation of the attenuated strain DENV-2 PDK-53in healthy adult volunteers was associated with high rates of seroconversion without serious adverse effects.  Chimeric viruses are produced by replacing prM and E genes of DENV-2 PDK-53 with that of DENV-1, DENV-3 and DENV-4. Since the mutations of DENV-2 PDK-53 that confer the attenuating phenotype to the virus lie in the non-structural protein encoding genes, replacement of prM and E genes did not result in alteration of the attenuating phenotype of the virus.
The intertypic chimera viruses showed a temperature-sensitive phenotype, reduced replication in mosquito cell lines, high degree of genetic stability and lack of neurovirulence in suckling mice.  Different tetravalent formulations containing chimeric DENV-1, DENV-3 and DENV-4 viruses along with DENV-2 PDK-53 were tested in NHPs. Subcutaneous injection of all formulations was well tolerated. Vaccination-induced neutralising antibodies to all serotypes after two doses with lower titres of anti-DENV-4 antibodies with some formulations.  A phase I clinical trial (registered at ClinicalTrials.gov, NCT01511250) of the combination of chimeric DENV-1, DENV-3 and DENV-4 with DENV-2 PDK-53, known by the name DENVax, was started by Inviragen Inc., Fort Collins, CO, USA in 2010 in healthy US volunteers.  The aim of the randomised double-blind placebo-controlled trial was to determine the dosage of each component of the vaccine for a balanced immune response in addition to safety and immunogenicity assessment. The results of the phase I trial are yet to be published. However, Inviragen has advanced DENVax to a phase II clinical trial. 
Dengue virus intetypic chimeras have also been generated using the cDNA backbone of the DEN4Δ30 attenuated strain developed through targeted mutagenesis by National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH). Replacement of prM and E genes of DEN4Δ30 by that of wild DENV-2 and DENV-3 generated the chimeric vaccine strains DEN 2/4Δ30 and DEN3/4Δ30, respectively. , This was done to mitigate the problem of lack of attenuation of DENV-2 and DENV-3 by Δ30 mutation in the UTR region. Both the vaccine candidates were genetically stable and showed low infectivity for mosquitoes. The growth rate in Vero cells was adequately high to facilitate production at a reasonable cost. The chimeric strains were safe and immunogenic at low doses of 10 3 pfu in humans. , DEN 1Δ30, DEN4Δ30, DEN 2/4Δ30 and DEN3/4Δ30 were combined to form a tetravalent formulation for testing in phase I clinical trial (registered at ClinicalTrials.gov, NCT01072786) in humans. 
Inactivated dengue vaccine
Cell culture-based inactivated whole dengue virus vaccine comprises of Vero cell grown DENV concentrated by ultracentrifugation followed by sucrose gradient-based purification and formalin inactivation. Purified, monovalent inactivated dengue vaccine developed at the Walter Reed Army Institute of Research (WRAIR), Maryland, USA, was shown to be safe and immunogenic in mice and Rhesus macaques. Three doses of inactivated vaccine protected the animals against challenge with a homologous wild DENV.  Three doses of inactivated monovalent dengue vaccine used along with adjuvants like alum or different combinations of aluminium hydroxide adsorbed on monophosphoryl lipid A induced high levels of neutralising antibodies in macaques.  Although inactivated vaccines are free from disadvantages like viral interference and reversion to a pathogenic phenotype, the role of these vaccines as the sole immunisation strategy is questionable because of conformational changes in viral proteins by formalin inactivation and lack of multiplication of inactivated viruses. However, inactivated dengue vaccine has been tested as the priming vaccine in a prime-boost immunisation strategy, with a live attenuated vaccine as the booster, leading to complete protection in macaques.  WRAIR, in collaboration with GlaxoSmithKline Biologicals and Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, is currently running a phase I trial of purified inactivated virus vaccine (registered at ClinicalTrials.gov, NCT01702857 and NCT01666652). 
Dengue subunit vaccine
Structural proteins of DENV like the envelope (E) glycoprotein that are capable of eliciting neutralising antibody responses form the basis of subunit dengue vaccines. Recombinant E protein antigen has been produced in various expression system like Escherichia More Details coli, viral vectors like baculovirus in insect cells, drosophila, vaccinia virus and in yeasts. ,, Domain III of E protein which contains numerous receptor-binding neutralising epitopes of DENV and induces serotype-specific antibodies is a potent subunit vaccine candidate. Removal of 20% of E protein at the carboxy-terminal, which codes for membrane anchor sequence, allows extracellular secretion and easy purification of this protein while retaining its antigenicity. The recombinant truncated E proteins, also known as r80E, of four DENV serotypes are being manufactured by Hawaii Biotech Inc., HI, USA and Merck and Co., NJ, USA. Low doses of r80E have been shown to induce complete protection in mice and monkeys as measured by PRNT.  The r80E DENV-1 subunit vaccine is currently undergoing human phase I clinical trial (registered at ClinicalTrials.gov, NCT01477580). 
Although subunit vaccines are safer than live attenuated vaccines owing to lack of multiplying virus in the body, they are poor immunogens thus requiring multiple booster doses and vaccine adjuvants which questions the cost-effectiveness of subunit vaccine candidates. Recently, a consensus DENV E protein domain III (cEDIII) comprising neutralising epitopes for all DENV serotypes has been constructed. This novel vaccine candidate in lipidated form (LcEDIII) was capable of inducing neutralising antibody response in mice. Since lipidated moieties activated antigen-presenting cells via Toll-like receptors, this vaccine did not require any exogenous adjuvants. 
Dengue DNA vaccine
DNA vaccines consist of antigen encoding genes cloned into a plasmid vector, which on inoculation is taken up antigen presenting cells (APCs). This leads intracellular generation of target antigens followed by their association with major histocompatibility complex (MHC) class I and induction of protective cytotoxic immune response. Early studies assessed the safety and immunogenicity of monovalent dengue DNA plasmid constructs expressing the truncated envelope (E) protein.  DNA vaccines expressing premembrane (prM) and full-length E proteins were shown to be immunogenic in NHPs who were partially protected against wild DENV challenge. Incorporation of prM was justified by the fact that prM acts as a chaperonin and helps in proper folding of E protein.  Subsequently chimeric DNA vaccines have been prepared by DNA shuffling and screening to express epitopes of the truncated E antigen that are shared by all four DENV serotypes. This vaccine was to mount neutralising antibody response and partial protection against multiple DENV serotypes in mice and Rhesus macaques. ,
Since DNA vaccines induce low levels of neutralising antibodies, strategies of incorporating intrinsic adjuvants into the plasmid vector like immunocostimulatory CpG motifs or granulocyte macrophage-colony stimulating factor are also being evaluated. , Recently, constructs of three proteins namely prM, E and NS-1 are also being tested in mice models.  Naval Medical Research Center, Walter Reed Army Institute of Research (WRAIR), Maryland, USA has developed a candidate dengue DNA vaccine by cloning prM and E of each serotype into plasmid vectors. In a proof of concept phase I trial of DENV-1 construct, the vaccine was well tolerated. Since it did not show adequate protective immune response it is being redesigned using a novel adjuvant. 
Virus vectored vaccines
Various viruses have been used as vectors for insertion of DENV genes and expression of DENV antigens, capable of eliciting neutralising antibody response. These include replication-defective adenovirus vectors, Venezuelan equine encephalitis (VEE) virus vector and attenuated measles virus (Schwarz vaccine strain). ,, Bivalent constructs expressing prM and E proteins from two serotypes each (DENV-1 and -3 in one and DENV-2 and -4 in another) have been prepared by insertion into an adenovirus vector (cAdVax). Inoculation of NHPs at separate sites lead to production of neutralising antibodies to respective DENV serotypes. , Subsequently, a tetravalent vaccine (cAdVax-DenTV) was prepared by mixing these constructs, which protected Rhesus macaques against DENV challenge by all serotypes.  Virus vectored vaccine can be used as a part of prime boosting strategy along with naked DNA vaccine. Prior immunity due to past adenovirus infection or exposure to the measles vaccine virus can limit replication and immune responses, which is less likely with VEE virus vectors.
Dengue vaccine candidates in different phases of clinical trial have been summarised in [Table 1].
|Table 1: Dengue vaccine approaches currently in different phases of clinical trial|
Click here to view
| ~ Prospect of Dengue Vaccine in India|| |
In the past decade, India has shown an increasing trend in the number of reported dengue cases, which have steadily increased from 5534 in 2007 to 75,454 in 2013 according to the National Vector Borne Disease Control Programme (NVBDCP).  With 18 out of 35 states of the country now being considered endemic for dengue and the spread of the disease from urban to suburban and rural areas, the actual number of cases may count in millions. With improvement in surveillance system and efficient management of cases, the case fatality rate due to severe dengue has declined from 3.3% in 1996 to 0.4% in 2010.  Although there has been a decline in dengue-associated mortality, hospitalisation rates due to severe dengue during outbreaks are on the rise. Due to repeated dengue outbreaks and epidemics, there has been a significant increase in the financial burden to the health care sector. This emphasises the need for a cost-effective dengue vaccine as preventive strategy, in conjunction with vector control. However, a number of issues need to be addressed.
A good community-based, hospital-based and laboratory-based surveillance system capable of monitoring the circulating DENV serotypes in different regions, is critical for a vaccine efficacy trial. In addition, for determination of vaccine efficacy, an active surveillance system capable of detecting all febrile illnesses due to dengue in the community, both mild and severe, is also necessary.  Thus a strong surveillance system needs to be in place before any vaccine trial takes place in endemic regions in India. This can be a challenge particularly in resource limited rural and suburban settings. In addition to DENV serotype-based epidemiological data, adequate data regarding the circulating genotypes of the virus are essential before vaccine implementation. This is because genotypic differences between the circulating strains and the vaccine strains, leading to antigenic mismatch, can result in poor vaccine efficacy as shown the ChimerVax trial in Thai school children, where a poor vaccine efficacy to DENV-2 was observed due to genotypic differences. , Another important consideration is that the safety, performance and cost-efficacy of the various vaccine candidates are not yet fully known. Since the vaccine efficacy trials have generated variable results in different endemic settings, it is not possible to predict the utility of a particular dengue vaccination strategy beforehand or to extrapolate the results of trials conducted elsewhere. In addition, the acceptable price of a new vaccine for usage in the public sector would vary from one country to another. To address the issue of cost-effective implementation, an economic analysis of any possible dengue vaccination strategy would need to be carried out in India.
A multiple dose regime, required for balanced immune response in dengue vaccination, can be difficult to implement in India because of the significant drop-out rates. Another important issue would be to fit a new vaccine approach in the already existing immunisation schedule. In a study published by Douglas et al. on the views of policy makers and opinion leaders from eight countries endemic for dengue, regarding usage of dengue vaccines in public sectors, government officials from India had shown an appropriately guarded approach towards the use of dengue vaccines in absence of adequate safety and efficacy data. However, expressed interests were high among non-government hospital officials and representatives of medical associations.  Successful implementation of a dengue vaccine in India will definitely require a involvement of both government and private sectors.
Indian scientists at the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi have developed a DENV-2 E protein based non-infectious virus-like particle (VLP) using the yeast Pitchia pastoris as the expression system.  The DENV-2 E VLP was able to induce high titre of neutralising antibodies in murine models. Since yeast expression system has the advantage of high yield, the VLP-based formulation can be an inexpensive vaccine for usage in resource-limited dengue endemic countries like India in future, once its efficacy and safety are established.
| ~ Conclusions|| |
In spite of the challenges confronted, the dengue vaccine development platform has progressed forward over the past few years, with a number of vaccine candidates in different phases of clinical trial. Evaluation of efficacy of the vaccine candidates has yielded variable results, a major reason being varying epidemiology of the disease in diverse populations. The situation may further be complicated by the recent discovery of a new serotype of DENV, distinct from serotypes 1-4, which was found to be circulating in macaques in Borneo, South-East Asia.  Sustained transmission of a new serotype in humans in the near future could become another obstacle in dengue vaccine development. Thus, an enhanced surveillance of the disease is required to generate detailed epidemiological data for different subsets of populations in different geographical locales before any dengue vaccine can be introduced into the field. Considering the potential risk of inducing immune enhancement, safety assessment of vaccines in clinical trials needs to be undertaken for longer period of time involving larger populations. In addition, there is an urgent need to develop and standardise improved immunodiagnostic assays for better prediction of the protective immune response in dengue. Lastly, regulations for dengue vaccine implementations should be formulated based on the needs of individual countries, avoiding competing interests.
The WHO and Pediatric Dengue Vaccine Initiative (PDVI) supported by Bill and Melinda Gates Foundation have been working together in collaboration with vaccine manufacturers and national regulatory bodies to accelerate dengue vaccine development and standardise field-testing of dengue vaccine.  The aim is to strengthen the dengue surveillance network, define the social and economic burden of dengue, identify trial sites and fund basic research towards making a safe and effective dengue vaccine a reality. In the near future dengue vaccination can become effective supplementary preventive measure to the existing vector control strategies to counteract the increasing global burden of dengue.
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