VIRAL IMMUNOLOGY Volume 28, Number 2, 201X ª Mary Ann Liebert, Inc. Pp. 1–9 DOI: 10.1089/vim.2014.0093

Dengue Vaccine Development: Strategies and Challenges Lakshmy Ramakrishnan,1,2 Madhavan Radhakrishna Pillai,1 and Radhakrishnan R. Nair1

Abstract

Infection with dengue virus may result in dengue fever or a more severe outcome, such as dengue hemorrhagic syndrome/shock. Dengue virus infection poses a threat to endemic regions for four reasons: the presence of four serotypes, each with the ability to cause a similar disease outcome, including fatality; difficulties related to vector control; the lack of specific treatment; and the nonavailability of a suitable vaccine. Vaccine development is considered challenging due to the severity of the disease observed in individuals who have acquired dengue-specific immunity, either passively or actively. Therefore, the presence of vaccine-induced immunity against a particular serotype may prime an individual to severe disease on exposure to dengue virus. Vaccine development strategies include live attenuated vaccines, chimeric, DNA-based, subunit, and inactivated vaccines. Each of the candidates is in various stages of preclinical and clinical development. Issues pertaining to selection pressures, viral interaction, and safety still need to be evaluated in order to induce a complete protective immune response against all four serotypes. This review highlights the various strategies that have been employed in vaccine development, and identifies the obstacles to producing a safe and effective vaccine.

internalized, the virion is taken up into endocytic vesicles, where the low-pH environment induces fusion of the envelope and the endosomal membranes, leading to the release of the infectious viral RNA into the cytoplasm, where viral replication and translation occur (2,3,67). The virus is transmitted to a human host through the bite of an infected mosquito, the primary vector being Aedes aegypti (35). These mosquitoes are highly domesticated, rest indoors, feed on humans during daylight hours, and prefer to lay their eggs in water-filled containers (34). Rapid population growth, unplanned urbanization, overcrowded housing facilities, poor sanitation, and the need for water storage have created areas that favor mosquito breeding. Ae. aegypti is densely populated in tropical and subtropical regions, and this correlates with DENV outbreaks in regions such as southeast Asia, the Indian subcontinent, Africa, and the Americas (94).

Dengue Virus

D

engue virus (DENV) is considered to be one of the most rapidly transmitting mosquito-borne viruses in the world, leaving approximately 2.5 billion people at risk of acquiring infection (94). The World Health Organization (WHO) estimates that 50–200 million dengue infections occur annually (94). Figure 1 depicts the geographic distribution of dengue-infected nations. DENV belongs to the family Flaviviridae, which comprises positive-sense singlestranded RNA viruses. The virus has four genetically distinct types: DENV-1, -2, -3, and -4. Serotypes 1 and 4 were observed to have sequence homology of 73%, serotypes 3 and 4 to have 54% homology, while serotype 2 was found to be considerably distinct from the other serotypes (10). The dengue virion is a 50 nm enveloped particle that adopts an icosahedral symmetry (66). The envelope is a lipid bilayer studded with two structural proteins—envelope (E) and membrane (M)—which have roles in host cell entry and virus maturation, respectively (39). Initial contact with the host cell is mediated by low affinity interactions between the E protein, cellular heparan sulfate groups, and glycosaminoglycans, concentrating virion particles onto the cell surface (17,90). The primary target of DENV is the immature dendritic cell, and entry into host cells is facilitated by the binding of E protein to its cognate receptor, dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) (87). Once 1 2

Disease Outcome

The WHO classified the clinical occurrences of DENV infection as dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) (94). Infected patients show a high, sustained fever for 2–7 days during the febrile phase, accompanied with headache, malaise, nausea, vomiting, myalgia, and abdominal pain. A diagnostic marker for DENV infection is a falling platelet count, where the levels may decrease from 250,000/lL to 100,000/lL over a couple of

Laboratory Medicine and Molecular Diagnostics, Rajiv Gandhi Centre for Biotechnology, Trivandrum, India. Department of Biotechnology, School of Life Sciences, Manipal University, Manipal, India.

1

2

RAMAKRISHNAN ET AL.

FIG. 1. Map of countries that have reported dengue virus infections. Source: World Health Organization Map Production: Public Health Information and Geographic Information Systems (GIS) World Health Organization; http://gamapserver .who.int/mapLibrary/Files/Maps/Global_DengueTransmission_ITHRiskMap.png days. Treatment is in the form of managing symptoms by administration of antipyretics and fluids. During defervescence, the patient may go into a critical phase, which is marked by an increase in capillary permeability, leading to a decrease in plasma volume. On adequate care and hospital attention, the patient can undergo circulatory and metabolic disturbances and enter a toxic stage, which lasts 24–48 h, before making a rapid recovery (18). The leaked fluid is reabsorbed once the vascular permeability resolves, platelet count increases, and the patient recovers. Patients whose condition improves after the fever are said to have nonsevere dengue infection. Progression to severe disease may occur if conditions deteriorate, and the WHO has formulated warning signs to enable clinicians to recognize patients who may enter into a toxic stage (94). Complications associated with DHF include dehydration and inadequate electrolyte intake, which can ultimately lead to pulmonary distress and sometimes death within 12–24 h after shock ensues. The loss of plasma may cause hypovolemia, which decreases cardiac output, leading to circulatory shock. In such cases, the administration of intravenous fluid to induce a rapid increase in blood pressure may become excessive and aggravate bleeding due to the sudden increase in blood flow. As a consequence, there is a volume overload leading to pulmonary edema, acute respiratory distress, and possible organ impairment (liver or kidney) (40). Despite the implementation of the DF/DHF/DSS classification system, there are cases where severe dengue infection can occur in the absence of any warning signs. Therefore, assessment also requires laboratory-confirmed diagnosis.

Immunopathogenesis of DENV Infection

Infection with any of the DENV serotypes gives rise to a similar clinical outcome. Individuals on a primary challenge with dengue appear to elicit immune responses that clear the virus and generate long-lasting protective immunity against secondary homotypic infections (47). The clinical manifestation is primarily DF under these circumstances. Re-exposure to the virus, with a different serotype (heterotypic infection), commonly results in DHF/ DSS, as the immune system is only transiently protected against the newly infecting serotype (72). Historically, this was observed in two populations: one was a group of infants (6–9 months old) whose maternal antibodies had not completely declined and who underwent a heterotypic infection; the other was a group of young children who had previously experienced febrile illness and who were exposed to a different DENV serotype (42). This indicates that pre-existing immunity to a viral serotype could aggravate disease during a heterotypic infection, as opposed to a more heightened protective immune response that would alleviate disease. There are several factors that come into play, resulting in a less than optimal immune response during DENV infection. Disarmament of the innate immune system

During the initial stages, the virus is able to disarm the innate immune system. Pattern recognition systems have the ability to shape the immune response on encounter with

DENGUE VACCINE DEVELOPMENT—SO NEAR YET SO FAR

pathogen. A strategy to avoid detection is the formation of isolated intracellular regions. DENV generates convoluted membranes, around which host and viral factors that are necessary for viral replication and translation localize (2). This region, within the cytosol, is remote, creating a microenvironment that is difficult for pattern recognition systems to detect. The ability of TLR-3 to aid in viral clearance has been emphasized in West Nile virus, by the antiviral activity of IFNa and IFN-b (23). Another study found that TLR-3 signaling also induced the production of IL-8, TNF-a, and TNF-b, suggesting that it plays a role in limiting DENV infection during the initial stages but the response may become detrimental if the pro-inflammatory response overrides its antiviral effect (88). Another mechanism to defer attack is by the action of the NS2B-NS3 protease complex, which acts as an antagonist of IFN production (69). Infected DCs are unable to mature and therefore unable to prime naı¨ve T cells effectively, preventing a proper immune bridge across innate and adaptive responses (15). Apoptosis of DCs was observed, in a manner similar to that seen in HCV infections, where the nonstructural proteins induced Fas ligand expression (54). In addition, some studies have shown that DENV-infected cells kill bystander cells in a mechanism comparable to that seen in Ebola virus (11). Collectively, these events influence the immune response, as it affects antigen availability.

3

fection progressing into DHF is prior exposure or pre-existing DENV immunity. Original antigenic sin

Individuals may encounter dengue on more than occasion during their lifetime, and it is likely that the newly infecting serotype will be different from the one previously encountered (42). The serotypes share 70% sequence homology. Therefore, to the immune system, the antigen appears considerably different to that previously encountered (10). The responding lymphocytes may have less than optimal avidity toward the epitopes expressed on the newly infecting virus. This alteration in the immune response due to the memory of the original antigen is referred to as original antigenic sin (25). The cross-reactive nature of the peptide enables it to bind to the T-cell epitope at certain anchoring positions (47). This partially activates certain T-cells but memory T-cells possess a lower threshold for antigen-stimulation compared to their naı¨ve counterparts and are readily activated. Moreover, antibodies generated during secondary infections respond better to the primary antigen, and this prevents the onset of a proper humoral response against the secondary antigen (25). Therefore, cross-reactive memory lymphocytes may be responsible for the altered cell behavior and responses.

Primary infections

The regulation of the adaptive immune response is crucial in determining whether infection results in viral clearance or a severe disease outcome. Primary DENV infections manifest as DF or are asymptomatic, due to a protective antiviral response, facilitated by IFN-c and TNF-a, that are generated by CD8 + T-cells (97). These responses are likely to be achieved in a CD4 + T-cell-independent manner, analogous to observations made on Listeria monocytogenes (83,97). Another protective shield is from the generation of neutralizing antibodies, preventing virus entry and membrane fusion events (73). Individuals who recalled previously being infected with DENV showed detectable levels of antiIgG, even after 60 years of exposure (47). These responses along with the antiviral cell-mediated response protect individuals from primary DENV challenge.

Cytokine storm phenomenon

Both serotype-specific and cross-reactive CD8 + T-cells have been detected in DENV-immune individuals. DENVspecific CD8 + T-cells have shown suboptimal degranulation accompanied with the generation of IFN-c, TNF-a, and IL-2, -4, and -6. This altered cytokine profile is referred to as a cytokine storm phenomenon. In addition, the CD4 + T-cell response is diverted from a Th1 cytokine profile to a Th2 profile, marked by the production of IL-6 and IL-10, similar to that observed during Leishmania infection (12). Similar T-cell responses have been observed in respiratory syncytial virus infections, contributing to enhanced pulmonary disease in mice models (6). These responses are not considered protective and are suggested to contribute to plasma leakage and endothelial cell damage (47).

Secondary infections and the onset of DHF

Antibody-dependent enhancement

A similar mechanism is thought to occur during homotypic infections, where T-helper type 1 (Th1) cytokines maintain the DENV-specific CD8 + T-cell population after an acute infection (97). Relatively higher levels of IFN-c and TNF-a were reported on a secondary DENV challenge, via DENV-specific CD4 + T-cells (61). Higher IFN-c levels were observed on homologous challenge compared to heterologous serotypes, explaining the long-lasting immunity during homotypic infections and the transient one imparted during heterotypic infections. Interestingly, the TNF-a level during heterologous responses was relatively stronger and broader, suggesting that it may have a role in the severe manifestations of secondary infections. The central challenge associated with dengue infection is the development of DHF/DSS. Progression to severe disease is influenced by factors such as the infecting serotype, viral virulence, viral load, host factors, and the nature of the immune response. The major risk factor of DENV in-

Antibody responses sparked an interest on observation of DHF/DSS in infants born to dengue-primed mothers who then underwent primary DENV infection (47). During heterotypic infections, the DENV-specific B-cells secrete subneutralizing antibodies (63). These antibodies associate with the virus to form antigen–antibody complexes, which are taken up by cells expressing FcR receptors. This uptake is referred to as antibody-dependent enhancement (ADE), and uptake occurs with more efficiency than free virus entry into host cells (42). ADE enhances the uptake of virions, leading to higher rates of viral replication, ultimately contributing to enhanced disease (70,79). Working in a similar fashion, immature virions have recently been considered as unveiled pathogens, with their ability to become internalized via antiprecursor membrane (prM) antibodies. Entry may lead to processing of their envelope layer, generating more fusogenic viruses, thereby increasing infectivity (99).

4 Complement pathway

The complement system is closely associated with the hemostatic system and responds to pathogen invasion. Dengue-infected patients have been observed to produce high levels of IgG1 and IgG3, which are effective activators of the complement cascade. The anti-NS1-NS1 complex induces the formation of the membrane attack complex. Increased levels of matrix metalloproteinase-2 and -9, decreased PECAM-1 (platelet/endothelial cell adhesion molecule-1) and vascular endothelium cadherin CAMS and the redistribution of F-actin fibers were suggested as contributors of vascular endothelial leakage in vitro (60). Predisposition

Host factors such as age, lifestyle, and genetic factors can also contribute to severe disease. Individuals who are considered to be at a high risk of acquiring DHF include infants younger than 1 year of age and those with underlying gastrointestinal bleeding and prolonged shock. Genetic factors such as TNF-a, transporter associated with antigen processing (TAP) polymorphism, and genetic variation in DC-SIGN may affect disease outcome: a single nucleotide substation in the promoter region of DC-SIGN may be associated with risk of a more severe disease outcome (84). The onset of DHF/DSS is therefore due to both host and viral factors that ultimately generate a detrimental immune response. Vaccine Development Strategies

Vaccination is the major preventative strategy against DENV, and the WHO has deemed vaccine development as a necessity (94). The main challenges to vaccine development include the presence of four serotypes, the risk of dengueprimed individuals developing severe disease on exposure to a different serotype, the lack of understanding of the pathophysiology of infection, and the lack of specific animal models. Surface regions of viral coats have previously been suggested as effective vaccine candidates due to the ability of their synthetic counterparts to generate specific cellular and humoral immunity. The E protein has been documented to induce heightened immune responses compared to the other structural and nonstructural proteins, and this is consistent with other flaviviruses. Several strategies have been implemented in the development of a vaccine against DENV. Live attenuated vaccines

Mahidol University, Thailand, was the first to develop monovalent live attenuated vaccines for each of the serotypes (8). Attenuation was achieved through serial tissue culture passage (41). Good immune responses were observed in the mono-, di-, tri-, and tetravalent preparations in American military and Thai volunteers (41,80). However, the need to assess clinical safety was highlighted when some participants experienced mild to moderate dengue-like systemic reactions (80). Selection pressures in vaccinations related to Poliomyelitis, Streptococcus pneumoniae, and Haemophilus influenzae have been reported (33,58). Taking into consideration the cocirculation of multiple dengue serotypes and the changing prevalence of serotypes during seasonal variations or within different geographical locations, targeted serotype vaccinations may lead to serotype replacement (33,58,59). This may

RAMAKRISHNAN ET AL.

occur in the form of an increase in the prevalence of the vaccine serotype or of the nonvaccine serotypes. Although vaccination would induce herd immunity, if serotype replacement occurs, then there would be an increase in the number of individuals exposed to serotypes that are not covered by vaccination. The major concern is the risk of acquiring DHF/DSS on natural exposure to DENV after immunization with a vaccine that does not cover all four serotypes (35). Several research teams proceeded with the tetravalent vaccine, obtaining similar results. Aventis Pasteur progressed to clinical trials and found that seroconversion of DENV-3 was more pronounced, suggesting that it had a replicative advantage (facilitation) in the tetravalent vaccine (51). The team at Walter Reed Army Institute of Research assessed the cytotoxic T-lymphocyte (CTL) response to this vaccine in a phase I clinical trial (78). Proliferative responses were higher to DENV-1 and -3, while cytolytic responses were greater to DENV-2 and -3. This suggests that the CTL responses are not equivalent to all the four serotypes. Sun et al. observed only 2 out of 10 participants seroconverted to all four serotypes after a single dose of the vaccine (86). More recently, Anderson et al. found that DENV-1 facilitated the seroconversion of DENV-3 and -4 but antagonized the response toward -2 (5). Another attenuation strategy is targeted mutagenesis within conserved 3¢UTR regions, which are implicated in viral replication (64). The recombinant vaccine is immunogenic and attenuated (93). Monovalent and tetravalent preparations were compared and good immune responses were reported (27). However, these vaccines may replicate at low levels within vaccinated individuals, raising concern over transmission to nonvaccinated individuals. Chimeric vaccines

Sanofi Pasteur have developed a novel tetravalent dengue vaccine candidate (CYD-TDV), based on the genetic backbone of a related flavivirus (37). The vaccine comprises of four recombinant live attenuated vaccines, expressing the DENV 1–4 prM-E region. The vaccine is considered to be safe and immunogenic, and a phase IIb trial found that it is efficacious and well tolerated (38,81). Primary findings found that all flavivirus-naı¨ve subjects underwent seroconversion after three doses of the vaccine preparation (36,68). Recently, a phase II study conducted in Singapore found that after three doses, only 66.5% of participants seroconverted to all four serotypes (56). Capeding et al. found the three-dose strategy to be efficacious in the Asia-Pacific region, but this phase III study did not take into consideration serotype-specific responses (13). One of the major concerns regarding live attenuated and chimeric vaccines is the composition of the vaccine: each tetravalent preparation is composed of four monovalent vaccines. Interaction between serotypes of differing viral fitness may lead to viral interference: competition and facilitation (5,28). Administration of multiple doses was implemented in several studies to insure complete immune protection. This has previously been implemented in oral polio vaccine (OPV), which was designed to protect against three polio serotypes. In the case of DENV infection, doses would pose a problem for vaccinated individuals living in dengue-endemic areas. The first 4–6 months after vaccination and before the second dose would be crucial, as immunity to heterotypic infections would persist, and it would be possible to acquire a heterotypic

DENGUE VACCINE DEVELOPMENT—SO NEAR YET SO FAR

infection placing the individual at risk of serious disease. Long-term evaluation of the safety of dosage strategies and examination of viral interactions would need to be studied to ensure clinical safety and efficacy. DNA vaccines

DNA-based vaccines previously faced difficulties in generating immune responses due to the low efficiency with which foreign DNA is taken up by host cells. Instead, some researchers turned to replication-deficient viral vectors expressing antigens de novo. A tetravalent vaccine was constructed using a pair of adenovirus vectors, expressing prM and E of two different serotypes (46). Humoral responses to all four serotypes have been reported in rhesus macaques and mice, but T-cell responses have not yet been studied (46,77). Men et al. constructed a highly attenuated replication-deficient modified vaccine Ankara (MVA) as a recombinant vector expressing a truncated version of the DENV-2 E protein (65). Monkeys immunized with the vectored vaccine were protected against challenge with DENV-2, suggesting investigations into putative tetravalent preparations. Baculovirus expression vectors have also been employed in murine studies but suffer from hindrances related to low expression levels and reduced immunogenicity (76). Vector immunity, especially to individuals previously exposed to adenovirus-based viral vector vaccines, and vector biosafety are concerns that need addressing (20,50). Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) are defective, nonpropagating virus-like particles that express a viral antigen. The advantage the VRP vaccine platform offers is its ability to target dendritic cells in the lymph nodes, facilitating antigen processing and presentation. The clinical safety of these alphavirus replicon particles has been assessed in phase I clinical trials in vaccines against HIV and CMV (92). A tetravalent VRP vaccine, expressing the ectodomain of DENV E protein, was able to induce neutralizing antibodies and T-cell responses to all the serotypes in an equivalent manner (52). Another advantage is its ability to overcome maternal antibody interference, as evidenced by a study where the VRP vaccine induced protective antibody responses in the presence of pre-existing maternal antibodies. This reinforces the potential of a dengue VRP vaccine for the infant population (91). The NS1 antigen is another candidate vaccine antigen that has been investigated. Mice injected with the plasmid pcTPANS1 encoding NS1 protein were found to produce high levels of anti-NS1 antibodies, and 100% survival was observed on challenge with DENV-2 (22). A subsequent study found that both humoral and cellular immune responses contribute to protection on virus challenge (96). The challenge associated with NS1-based vaccines, however, is the risk of anti-NS1 antibodies cross-reacting with endothelial cells and human platelets, contributing to endothelial cell and platelet damage and inflammation (57). Chen et al. constructed an NS1 protein lacking a region of the C-terminal domain that was found to contain cross-reactive epitopes and designated it as DC NS1 (16). Platelet dysfunction and bleeding were observed only in mice challenged with the anti-full-length NS1 and not with anti-DC NS1 antibody. These studies suggest further investigation into the development of a NS1-based vaccine is necessary, taking into consideration that NS1 induces serotype-specific immune responses.

5

Subunit vaccines

Subunit vaccines possess a higher safety profile compared to whole virus vaccines, but they do not offer any immunological advantage. A mammalian cell line was engineered to produce extracellular particles (EPs) containing DENV-2 E protein, which were found to be immunogenic in mice (53). A follow-up study had similar observations but utilized the Sf9 insect system to develop antigenic EPs (55). In an attempt to enhance the immunogenicity of subunit viral particles and achieve higher expression levels, studies have turned to recombinant subunit systems. Truncated recombinant E protein subunits (80E) expressed from Drosophila Schneider-2 (S2) cells from each of the serotypes, combined with ISCOMATRIX adjuvant, was found to induce neutralizing antibody responses in mice and in nonhuman primates (19). Phase I clinical trials of this DEN-80E vaccine are currently underway (21). Inactivated vaccines

Inactivated vaccines do not represent the virus as a whole but are sufficiently immunogenic. The advantages of using inactivated vaccines are the elimination of reversion of the virus to a more pathogenic phenotype and the reduction in viral interaction. A study on a JEV-inactivated vaccine found that it could induce both a humoral and a cell-mediated immune response, suggesting a similar possibility to occur in DENV (1). In 2012, Fernandez et al. reported that the dengue purified inactivated vaccine (DPIV) with adjuvant AS03A was immunogenic in rhesus macaques, and they have proceeded to carry out phase I studies in humans (30). Future Vaccine Development Considerations

The main challenge in development is in offering protection to all four serotypes. The function of the protein remains the same despite the amino acid sequence variation in the E protein between the DENV serotypes. This suggests that functionality can constrain the extent of diversity of the antigen, implying that these essential regions may contain conserved sequences, which can act as effective viral epitopes in the construction of vaccines (29). The idea of selecting conserved protein sequences among different viral strains has been previously described in Influenza A, where current vaccines do not provide protection against seasonal or re-emerging variants (43,85). Hemagglutinin, the fusion peptide, was found to contain conserved regions in its Nterminus, which induced immune responses and offered protection on rechallenge with different subtypes (49). Therefore, an ideal vaccine candidate against DENV would be a region whose function is conserved across the existing strains. Ramanathan et al. have developed a universal vaccine construct, DNA SynCon, which was found to induce a humoral response in mice (75). The construct consists of a consensus region from domain III of the E protein from all four serotypes, which was cloned into a mammalian expression vector. The safety and immunogenicity of this candidate needs to be further evaluated. Six conserved regions across the four serotypes within the E protein were identified as putative epitopes on a study looking into DENV isolates from South India (74). Testing for immunogenicity is required to assess these epitopes further. More

6

recently, Mani et al. on taking lessons from the HPV and HBSAg vaccines have developed DENV-2 VLPS (62). These particles were found to be immunogenic but produced modest antibody titers. Further studies could investigate conserved epitopes, carry out codon optimization, and develop VLPs as putative vaccine candidates. This approach may resolve issues relating to serotype coverage and doses, allowing for simultaneous protection over all four serotypes. Demand for a dengue vaccine

Taking into consideration the various vaccine studies that are currently underway, a licensed dengue vaccine appears to be on the horizon. Several measures would need to be undertaken to ensure that the government, healthcare systems, and public and private sectors are prepared to meet the demands for a dengue vaccine, particularly in endemic regions (4). The Dengue Vaccine Initiative (DVI) is a consortium of organizations, comprising the International Vaccine Institute, the WHO, the Sabin Vaccine Institute, and the International Vaccine Access Center. These organizations have teamed up to encourage the development of dengue vaccines and aid in the formation of policies based on evidence-based research. The prevention boards formed by the DVI, Asia-Pacific Dengue Prevention Board, and the Americas Dengue Prevention Board recently held meetings to discuss plans to introduce a dengue vaccine. These meetings highlighted the need for the development of policies relating to vaccine safety and efficacy, logistics, surveillance data, vector control measures, and the involvement of the private and public sector in supporting the community (24). The demand for dengue vaccines was assessed by conducting surveys with policy makers and scientific professionals in eight endemic countries, covering Asia (India, Sri Lanka, Thailand, and Vietnam) and Latin America (Brazil, Colombia, Mexico, and Nicaragua) (26). The implementation of dengue vaccines was found to be favorable with the major determinants of use being WHO recommendation, economical pricing, introduction into regular childhood immunization schedules, and the availability of additional funding to lowerincome endemic countries. The prospect of a safe and effective vaccine becoming available in the near future accelerated the formation of the Mexico Dengue Expert Group (MDEG), which was formed as an ad hoc think-tank unit, comprising scientists, academicians, public healthcare providers, the private sector, and public health officials (7). The burden of dengue infections on the healthcare system in Mexico, as evidenced by an increase in the incidence of DF from 1.7 cases per 100,000 inhabitants in 2000 to 43.03 cases per 100,000 inhabitants in 2012, is alarming (7). MDEG took into consideration the adoption of a vaccine, which required analysis of scientific studies and clinical trials, and the development of policies relating to immunization schedules. The analysis framework, developed by MDEG, has taken into consideration political and technical issues pertaining to the introduction of a dengue vaccine and has strengthened Mexico’s capacity for innovations in public health (7). Vector control strategies

Control of dengue infection through vaccination would need to be complemented with effective vector control strategies. Vector control measures have been implemented

RAMAKRISHNAN ET AL.

as a means of limiting dengue transmission and interrupting human–vector contact. Environmental management

Environmental management aims to reduce mosquito propagation by modifying mosquito habitat regions and minimizing human–vector contact (95). Water supply and storage systems may be improved by installing piped water to households, reducing larval habitats. Water-storage containers, collected during rainwater harvesting, can be fitted with meshes to prevent mosquito entry. Street cleansing systems would be useful in removing stagnant water, which serves as a breeding ground for urban pests. Long-term approaches include modifications to the infrastructure, such as installation of mosquito screens on doors and windows, repairing cement drains, and discouraging the construction of roof gutters on buildings (14). Chemical control

Larvicides have been used as a means to lower vector density in larval habitats, such as water-storage containers. These chemicals are considered to be of low toxicity and safe to use, and have been reported to be an effective intervention in Cambodia during seasonal outbreaks (14). Alternatively, insecticides targeting adult vectors directly influence transmission rates. Space spraying is recommended during emergencies where outbreaks require a massive reduction in vector density (14). Biological control

Dengue may be controlled through the introduction of organisms that prey upon, compete with, or parasitize Aedes mosquitoes, reducing their population (95). These biological control agents target the immature larval stages of the mosquito vector and are therefore distributed into waterstorage containers that serve as breeding sites. Studies in Vietnam have reported the effectiveness of Mesocyclops, a cyclopoid copepod—crustaceans that breed on first instar mosquito larvae—as effective control agents (71). A study in Cambodia found a significant reduction in Aedes infestation when the larvivorous fish, Poecilia reticulate, was distributed into water-filled containers and tanks (82). An innovative strategy in vector control is in utilizing the symbiotic relationship between Wolbachia bacteria and insects. In the context of disease, the bacterium is able to protect the mosquito vector from various pathogens, thereby limiting their transmission to humans (48). Wolbachia is transfected into Ae. aegypti, as it is naturally free from the bacterium, and holds the advantage in its ability to propagate through a population by inducing cytoplasmic incompatibility within the host (98). Transfection of Ae. aegypti with wMel and wMelPop-CLA Wolbachia strains resulted in a life-span-shortening phenotype and reduced transmission of DENV (44,45,89). Mosquitoes with wMel were released in the field in Cairns, Australia, in 2011, and it has been reported that these mosquitoes showed reduced DENV replication within the body and limited dissemination to the head region, consistent with previous reports that Wolbachia inhibits DENV replication and transmission in the mosquito vector (9,32). A subsequent study found that these infections are stable and displayed cytoplasmic incompatibility.

DENGUE VACCINE DEVELOPMENT—SO NEAR YET SO FAR

These studies suggest the implementation of Wolbachia-based biocontrol strategies in controlling dengue. Another approach carried out by Franz et al. was to develop Ae. aeypti exhibiting impaired vector competence for DENV-2 by triggering a natural RNA interference pathway within the mosquito midgut (31). This approach may also be utilized as a dengue control tool. Conclusions

The main drawbacks of current vaccine candidates are the possibility of viral interference, increasing disease susceptibility in vaccinated individuals, thereby negating any positive effects of vaccination. In addition, dosing strategies would also be problematic, as DENV serotypes co-circulate within a given geographical location. Issues relating to immune responses to all serotypes in an equivalent manner and vaccine safety also need to be addressed. Steps toward developing a universal DENV vaccine can be carried out by studying conserved antigenic regions. Lessons from other vaccine studies would also be useful. Taking into consideration the challenges associated with selection pressures, viral interaction, and dose strategies, a vaccine that offers complete serotype coverage is still the need of the hour. Author Disclosure Statement

No competing financial interests exist. References

1. Aihara H, Takasaki T, Toyosaki-Maeda T, et al. T-cell activation and induction of antibodies and memory T cells by immunisation with inactivated Japanese encephalitis vaccine. Viral Immunol 2000;13:179–186. 2. Alcaraz-Estrada SL, Yocupicio-Monroy M, and del Angel RM. Insights into dengue virus replication. Future Virol 2010;5:575–592. 3. Alen MMF, and Schols D. Dengue virus entry as target for antiviral therapy. J Trop Med 2012;2012:1–13. 4. Amarasinghe A, Wichmann O, Margolis HS, et al. Forecasting dengue vaccine demand in disease endemic and non-endemic countries. Hum Vaccines 2010;6:745–753. 5. Anderson KB, Gibbons RV, Edelman R, et al. Interference and facilitation between dengue serotypes in a tetravalent live dengue virus vaccine candidate. J Infect Dis 2011;204: 442–450. 6. Bangham CRM, Cannon MJ, Karzon DT, et al. Cytotoxic T cell response to respiratory syncytial virus in mice. J Virol 1985;56:55–59. 7. Betancourt-Cravioto M, Kuri-Morales P, GonzalezRoldan JF, et al. Introducing a dengue vaccine to Mexico: development of a system for evidence-based public policy recommendations. PLoS Negl Trop Dis 2014;8:e3009. 8. Bhamarapravati N, and Sutee Y. Live attenuated tetravalent dengue vaccine. Vaccine 2000;18:44–47. 9. Bian G, Xu Y, Lu P, et al. The endosymbiotic bacterium Wolbachia induces resistance to dengue virus in Aedes aegypti. PLoS Pathog 2010;6:e1000833. 10. Blok J. Genetic relationships of the dengue virus serotypes. J Gen Virol 1985;66:1323–1325. 11. Bray M, and Geisbet TW. Ebola virus: the role of macrophages and dendritic cells in the pathogenesis of Ebola haemorrhagic fever. Int J Biochem Cell Biol 2005;8:1560–1568.

7

12. Cann AJ. Pathogenesis. In: Principles of Molecular Virology. 5th ed. San Diego, CA: Elsevier Academic Press, 2005:208–248. 13. Capeding MR, Tran NH, Hadinegoro DR, et al. Clinical Efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observermasked, placebo-controlled trial. Lancet 2014;384:1358– 1365. 14. Chang MS, Christophel EM, Gopinath D, et al. Challenges and future perspective for dengue vector control in the Western Pacific Region. Western Pac Surveill Response J 2011;2:9–16. 15. Chase AJ, Medina FA, and Munoz-Jordan JL. Impairment of CD4 + T cell polarisation by dengue virus-infected dendritic cells. J Infect Dis 2011;203:1763–1774. 16. Chen MC, Lin CF, Lei HY, et al. Deletion of the Cterminal region of dengue virus nonstructural protein 1 (NS1) abolishes anti-NS1-mediated platelet dysfunction and bleeding tendency. J Virol 2009;183:1797–1803. 17. Chen Y, Maguire T, and Marks RM. Demonstration of binding of dengue virus envelope protein to target cells. J Virol 1996;70:8765–8772. 18. Chuansumrit A, and Tangnararatchakit K. Pathophysiology and management of dengue haemorrhagic fever. Transfus Altern Transfus Med 2006;8:3–11. 19. Clements DE, Coller BA, Lieberman MM, et al. Development of a recombinant tetravalent dengue virus vaccine: immunogenicity and efficacy studies in mice and monkeys. Vaccine 2010;28:2705–2715. 20. Cohen P. Immunity’s yin and yang. International AIDS Vaccine Report 2006;10. 21. Coller BG, Clements DE, Bett AJ, et al. The development of recombinant subunit envelope-based vaccines to protect against dengue virus induced disease. Vaccine 2011;29: 7267–7275. 22. Costa SM, Freire MS, and Alves AMB. DNA vaccine against the non-structural 1 protein (NS1) of dengue 2 virus. Vaccine 2006;24:4562–4564. 23. Daffis S, Samuel MA, Suthar MS, et al. Toll-like receptor 3 has a protective role against West Nile virus infection. J Virol 2008;82:10349–10358. 24. Dengue Vaccine Initiative: Dengue Prevention Boards 2014. Available at www.denguevaccine.org/consortium (accessed October 1, 2014). 25. de St Groth SF, and Webster RG. Disquisitions on original antigenic sin. I Evidence in man. J Exp Med 1966;124: 331–345. 26. Douglas DL, DeRoeck DA, Mahoney RT, et al. Will dengue vaccines be used in the public sector and if so, how? Findings from an 8-country survey of policymakers and opinion leaders. PLoS Negl Trop Dis 2013;7:e2127. 27. Durbin AP, Whitehead SS, Shaffer D, et al. A single dose of the DENV-1 candidate vaccine rDEN1D30 is strongly immunogenic and induces resistance to a second dose in a randomised trial. PLoS Negl Trop Dis 2011;5:1–10. 28. Edelman R. Unique challenges faced by the clinical evaluation of dengue vaccines. Exp Rev Vaccines 2011;10:133–136. 29. Ekiert DC, and Wilson IA. Broadly neutralising antibodies against influenza virus and prospects for universal therapies. Curr Opin Virol 2012;2:134–141. 30. Fernandez S. Immunogenicity and protection elicited by adjuvanted tetravalent dengue virus purified inactivated vaccine (TDENV PIV) in rhesus macaques. ASTMH 61st Annual Meeting, November 11–15, 2012, Atlanta, GA.

8

31. Franz AWE, Sanchez-Vargas I, Adelman ZN, et al. Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. Proc Natl Acad Sci U S A 2006;103:4198–4203. 32. Frentiu FD, Zakir T, Walker T, et al. Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia. PLoS Negl Trop Dis 2013;8:e2688. 33. Griffin DE. Emergence and re-emergence of viral diseases of the central nervous system. Prog Neurobiol 2010;91:95–101. 34. Gubler DJ. Dengue, urbanisation and globalization: the unholy trinity of the 21st century. Trop Med Health 2011;39:3–11. 35. Gubler DJ. Epidemic dengue/dengue haemorrhagic fever: a global public health problem in the 21st century. Dengue Bull 1997;21:1–19. 36. Guy B, Guirakhoo F, Barban V, et al. Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses. Vaccine 2010;8:632–649. 37. Guy B, Saville M, and Lang J. Development of Sanofi Pasteur tetravalent dengue vaccine. Hum Vaccin 2010;6: 696–705. 38. Guy B. Immunogenicity of Sanofi Pasteur tetravalent dengue vaccine. J Clin Virol 2009;46:S16–S19. 39. Guzman MG, Halstead SB, Artsob H, et al. Dengue: a continuing global threat. Nature Rev Microbiol 2010;8:7–16. 40. Hall JE. The body fluids and kidneys. In: Guyton and Hall Textbook of Medical Physiology. 12th ed. Philadelphia, PA: Saunders, 2011:285–300. 41. Halstead SB, and Marchette NJ. Biologic properties of dengue viruses following serial passage in primary dog kidney lines: studies at the University of Hawaii. Am J Trop Med Hyg 2003;69:5–11. 42. Halstead SB. Observations related to pathogenesis of dengue haemorrhagic fever. Yale J Biol Med 1969;42:350–362. 43. Heiny A, Miotto O, Srinivasan KN, et al. Evolutionarily conserved protein sequences of influenza A viruses, avian and human, as vaccine targets. PLoS One 2007;2:1–14. 44. Hoffmann AA, Iturbe-Ormaetxe I, Callahan AG, et al. Stability of the wMel Wolbachia infection following invasion into the Aedes aegypti populations. PLoS Negl Trop Dis 2014;8:e3115. 45. Hoffmann AA, Montgomery BL, Popovichi J, et al. Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission. Nature 2011;476:454–457. 46. Holman DH, Wang D, Raviprakash K, et al. Two complex, adenovirus-based vaccines that together induce immune responses to all four dengue virus serotypes. Clin Vaccine Immunol 2007;14:182–189. 47. Imrie A, Meeks J, Gurary A, et al. Antibody to dengue 1 detected more than 60 years after infection. Viral Immunol 2007;20:672–675. 48. Iturbe-Ormaetxe I, Walker T, and O’Neill SL. Wolbachia and biological control of mosquito-borne disease. EMBO Rep 2011;12:508–518. 49. Janice H, Akerstrom S, Shen S, et al. An antibody against a novel and conserved epitope in haemagglutinin 1 subunit neutralises numerous H5N1 influenza viruses. J Virol 2010;84:8275–8286. 50. Jechlinger W. Optimisation and delivery of plasmid DNA for vaccination. Exp Rev Vaccines 2006;5:803–825. 51. Kanesa-thasan N, Sun W, Kim-Ahn G, et al. Safety and immunogenicity of attenuated dengue vaccines (Aventis Pasteur) in human volunteers. Vaccine 2001;19:3179–3188.

RAMAKRISHNAN ET AL.

52. Khali SM, Tonkin DR, Mattocks MD, et al. A tetravalent alphavirus-vector based dengue vaccine provides effective immunity in an early life mouse model. Vaccine 2014;32: 4068–4074. 53. Konishi E, and Fujii A. Dengue type 2 virus subviral extracellular particles produced by a stably transfected mammalian cell line and their evaluation for a subunit vaccine. Vaccine 2002;20:1058–1067. 54. Krishnadas DK, Ahn JS, Han J, et al. Immunomodulation by hepatitis c virus-derived proteins: targeting human dendritic cells by multiple mechanisms. Int Immunol 2010;22:491–502. 55. Kuwahara M, and Konishi E. Evaluation of extracellular subviral particles of dengue virus type 2 and Japanese encephalitis virus produced by Spodoptera frugiperda cells for use as vaccine and diagnostic antigens. Clin Vaccine Immunol 2010;17:1560–1566. 56. Leo YS, Wilder-Smith A, Archuleta D, et al. Immunogenicity and safety of recombinant tetravalent dengue vaccine (CYD-TDV) in individuals aged 2–45 years Phase II randomised controlled trial in Singapore. Hum Vaccin Immunother 2012;8:1259–1271. 57. Lin CF, Wan SW, Cheng HJ, et al. Autoimmune pathogenesis in dengue virus infection. Viral Immunol 2006;19: 127–132. 58. Lipstitch M. Bacterial vaccines and serotype replacement: lessons from Haemophilius influenza and prospects for Streptococcous pneumonia. Emerg Infect Dis 1999;5:336–345. 59. Lipstitch M. Vaccination against colonising bacteria with multiple serotypes. Proc Natl Acad Sci U S A 1997;94: 6571–6576. 60. Luplerdlop N, Misse D, Bray D, et al. Dengue-virus-infected dendritic cells trigger vascular leakage through metalloproteinase overproduction. EMBO Rep 2006;7:1176–1181. 61. Mangada MM, and Rothman AL. Altered cytokine responses of dengue-specific CD4 + T cells to heterologous serotypes. J Immunol 2005;175:2676–2683. 62. Mani S, Tripathi L, Raut R, et al. Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titre neutralising antibodies. PLoS One 2013;8:1–9. 63. Mathew A, West K, Kalayanarooj S, et al. B-cell responses during primary and secondary infections in humans. J Infect Dis 2005;204:1514–1522. 64. Men R, Bray M, Clark D, et al. Dengue type 4 virus mutants containing deletions in the 3¢ noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 1996;70:3930–3937. 65. Men R, Wyatt L, Tokimatsu I, et al. Immunisation of rhesus monkeys with a recombinant of modified vaccina virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge. Vaccine 2000;18:3113–3122. 66. Modis Y, Ogata S, Clements D, et al. A ligand binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci U S A 2003;100:6986–6991. 67. Modis Y, Ogata S, Clements D, et al. Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein. J Virol 2005;79:1223–1231. 68. Morrison D, Legg TL, Billings CW, et al. A novel tetravalent dengue vaccine is well tolerated and immunogenic against all 4 serotypes in Flavivirus-naı¨ve adults. J Infect Dis 2009;201:370–377.

DENGUE VACCINE DEVELOPMENT—SO NEAR YET SO FAR

69. Morrison J, Aguirre S, and Fernandez-Sesma A. Innate immunity evasion by dengue virus. Viruses 2012;4:397–413. 70. Murphy BR, and Whitehead SS. Immune response to dengue virus and prospects for a vaccine. Annu Rev Immunol 2011;29:587–619. 71. Nam VS, Yen NT, Kay BH, et al. Eradication of Aedes aegytpi from a village in Vietnam, using copepods and community participation. Am J Trop Med Hyg 1998;59: 657–660. 72. Nisalak A, Endy TP, Nimmannitya S, et al. Serotypespecific dengue virus circulation and dengue disease in Bangkok, Thailand from 1973–1999. Am J Trop Med Hyg 2003;68:191–202. 73. Pierson TC, and Diamond MS. Molecular mechanisms of antibody-mediated neutralisation of flavivirus infection. Expert Rev Mol Med 2009;10:1–17. 74. Ramakrishnan L. ‘‘Novel vaccine candidate that offers protection over all four dengue serotypes’’, (Master of Science thesis, Manipal University, Manipal, Karnataka, India, 2013). 75. Ramanathan MP, Kuo Y, Selling BH, et al. Development of a novel DNA SynCon tetravalent dengue vaccine that elicits immune responses against four serotypes. Vaccine 2009;29:6444–6447. 76. Rantam FA, Purwati, Soegijanto S, et al. Analysis of recombinant, multivalent dengue virus containing envelope (E) proteins from serotypes - 1, - 3, and - 4 and expressed in baculovirus. Trials Vaccinol 2013; doi: 10.1016/j.trivac .2013.10.001. 77. Raviprakash K, Wang D, Ewing D, et al. A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus. J Virol 2008;82:6927– 6934. 78. Rothman AL, Kanesa-thasan N, West K, et al. Induction of T-lymphocyte responses to dengue virus by a candidate tetravalent live attenuated virus vaccine. Vaccine 2001; 19:4694–4699. 79. Rothman AL. Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms. Nature Rev Immunol 2011;11:532–543. 80. Sabchareon A, Lang J, Chanthavanich P, et al. Safety and immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role of serotype concentration, ratio, and multiple doses. Am J Trop Med Hyg 2002;66:264–272. 81. Sabchareon A. Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial. Lancet 2012;380:1559–1567. 82. Seng CM, Setha T, Nealon J, et al. Community-based use of the larvivous fish Poecilia reticulata to control dengue vector Aedes aegypti in domestic water storage containers in rural Cambodia. J Vector Ecol 2008;33:139–144. 83. Shedlock DJ, Whitmire JK, Tan J, et al. Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection. J Immunol 2003;170: 2053–2063. 84. Soundravally S, and Hoti SL. Significance of transporter associated with antigen processing 2 (TAP2) gene polymorphisms in susceptibility to dengue viral infection. J Clin Immunol 2008;28:256–262. 85. Stanekova Z, and Vareckova E. Conserved epitopes of influenza a virus including protective immunity and their

86.

87. 88. 89. 90.

91.

92.

93.

94.

95.

96.

97. 98. 99.

9

prospects for universal vaccine development. Virol J 2010; 7:1–13. Sun W, Edelman R, Kanesa-Thasan N, et al. Vaccination of human volunteers with monovalent and tetravalent liveattenuated dengue vaccine candidates. Am J Trop Med Hyg 2003;69:24–31. Tassaneetrithep B, Burgess TH, Granelli-Piperno A, et al. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med 2004;197:823–829. Tsai Y, Chang S, Lee C, et al. Human TLR3 recognises dengue virus and modulates viral replication in vitro. Cell Microbiol 2009;11:604–615. Walker T, Johnson PH, Moreira LA, et al. The wMel Wolbachia strain blocks dengue and invades caged Aedes aegpti. Nature 2011;476:450–453. Watterson D, Kobe B, and Young PR. Residues in domain III of dengue virus envelope glycoprotein involved in cellsurface glycosaminoglycan binding. J Gen Virol 2012; 93:73–83. White LJ, Parsons MM, Whitmore AC, et al. An immunogenic and protective alphavirus replicon particle-based dengue vaccine overcomes maternal antibody interference in weaning mice. J Virol 2007;81:10329–10339. White LJ, Sariol CA, Mattocks MD, et al. An alphavirus based-vector tetravalent dengue virus vaccine induces a rapid protective immune response in macaques that differs qualitatively from immunity induced by live virus infection. J Virol 2013;87:3409–3424. Whitehead SS, Falgout B, Hanley KA, et al. A live, attenuated dengue virus type 1 vaccine candidate with a 30nucleotide deletion in the 3¢ untranslated region is highly attenuated and immunogenic in monkeys. J Virol 2003; 77:1653–1657. World Health Organization. Dengue Guidelines for Diagnosis, Treatment, Prevention, and Control 2009. Available at www.who.int/rpc/guidelines/9789241547871/ en/ (accessed April 15, 2013). World Health Organization. Global Strategy for Dengue Prevention and Control 2012–2020. Available at www .who.int/denguecontrol/control_strategies/en/ (accessed October 1, 2014). Wu SF, Liao CL, Lin YL, et al. Evaluation of protective efficacy and immune mechanisms of using a non-structural protein NS1 in DNA vaccine against dengue 2 virus in mice. Vaccine 2003;21:3919–3929. Yauch LE, Zellweger RM, Kotturi MF, et al. A protective role for dengue virus specific CD8 + T cells. J Immunol 2009;182:4865–4873. Ye YH, Woolfit M, Rances E, et al. Wolbachia-associated bacterial protection in the mosquito Aedes aegypti. PLoS Negl Trop Dis 2013;7:e2362. Zybert IA, van der Ende-Metselaar H, Wilschut J, et al. Functional importance of dengue virus maturation: infectious properties of immature virions. J Gen Virol 2008; 89:3047–3051.

Address correspondence to: Dr. Radhakrishnan R. Nair Rajiv Gandhi Centre for Biotechnology Trivandrum Kerala 695 014 India E-mail: [email protected]