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Dengue is a disease of humans characterized by mild to severe fever leading to sometimes fatal outcomes (15). It is caused by Dengue virus (DV) which is a member of the family Flaviviridae, genus Flavivirus and exists as four closely related but antigenically distinct serotypes, DENV- 1-4. The virus is transmitted through blood following the bite of two species of Aedes mosquito, Aedes aegypti and A. albopicus. A. albopicus is an inefficient vector producing a slow moving outbreak when compared to A. aegypti, which produces sharp epidemics (18). Infection with any of the four serotypes of DV causes mild dengue fever (DF) to severe dengue haemorrhagic fever (DHF) and fatal dengue shock syndrome (DSS) (26). Dengue haemorrhagic fever (DHF) and DSS is primarily a disease found to affect children under the age of 15 in hyper- endemic areas in which all four serotypes of DENV are circulating (26). Dengue viruses are mostly found in the tropical and sub-tropical countries, putting nearly two-fifths of the world population at risk of infection. Epidemic dengue infection is currently documented in over 100 countries with recent isolation revealing more virulent strains of dengue viruses (10). The growing incidences of dengue is attributed to the increase in mosquito population, urbanization, travel and the difficulty in sustaining effective vector control measures over a long period of time (22). Since there is no specific treatment for dengue diseases and preventive measures presently rely on vector control and personal protection measures, it is necessary to develop a safe and effective vaccine. The DV is a positive strand RNA of about 11Kb and produces three structural proteins [Capsid (C), pre-membrane or membrane (prM), and envelope (E)] and seven non-structural proteins (NS-1, 2A, 2B, 3, 4A, 4B and 5) (6). The pathogenesis of DV and mechanism of different immune responses towards protection and/or disease is poorly understood and this poses significant challenges to development of vaccine against DV (23). Dengue virus infection also elicits significant cellular immune responses, a majority of which is directed against non-structural proteins. The role of antibodies to non-structural protein NS1, as well as the role of cellular immune responses in the course of the disease is not clearly understood (15).
The four antigenically distinct serotypes of DV elicit homologous immune responses to each of its own type. The epidemiologic observation that severe dengue disease (DHF/DSS) is more often associated with secondary dengue infections (16) has led to the hypothesis of antibody dependent enhancement (ADE) of DV infection in which antibodies from a primary infection bind but do not neutralize the virus during a secondary infection, and the virus-antibody complex gains entry into target cells via the Fc receptor (15). The adaptive immune response to DENV infection contributes to the resolution of infection and has a major role in protection from re-infection. Conversely, it is also believed to have a crucial role in the enhancement of disease severity seen in patients with DHF or DSS. The presence of neutralizing antibodies directed against the virus envelope (E) protein is the main mediator of protection against DENV infection, and induction of protective levels of neutralizing antibodies is therefore the major goal of immunization. Both live attenuated vaccines and non-living vaccines, such as inactivated virus vaccines, virus-like particles or DNA vaccines, each readily induce both neutralizing antibodies and protective immunity. Robust neutralizing antibody responses develop after DENV infection and are believed to provide lifelong protection against re-infection with the same DENV serotype and short lived protection of only a few months duration against a heterologous DENV serotype. This short period of cross-protection has been associated with the presence of cross-reactive neutralizing antibodies that wane rapidly after infection, though the exact mediator of this protection has not been identified. Another feature of immunity to DENV is the ability of a second administration of a live attenuated, tetravalent DENV vaccine to infect monkeys in which neutralizing antibodies have been induced by the dose of vaccine. The boosting effect is seen when second dose is given after a long interval it is postulated that the ability of DENV to infect using the IgG Fc receptor (FcR) allows it to infect a sufficient number of cells in the presence of neutralizing IgG antibody to induce a vigorous secondary immune response. It is possible that the boost in titer to all four DENV serotypes is the result of a breakthrough infection by a single serotype that induces a heterotypic boost in neutralizing antibody to all four serotypes (15).
An ideal DENV vaccine should be free from significant reactogenecity, should induce the level of protection afforded by infection with any of the four wild-type viruses, and provide lifelong protection while being economical. Live attenuated vaccines have been favoured to prepare DV vaccines. A potential drawback of deriving attenuated viruses by passage in cell culture is the accumulation of random, unintended mutations in antigen coding sequence which could lead to reduced immunogenecity. This problem could be addressed by employing full length infectious clones of DV virus genomes and attenuate these by introducing targeted deletions in the 3ââ‚¬â„¢- untranslated region of the genome by site directed mutagenesis. These techniques have also allowed for preparation of chimeras used for vaccine preparation. One of the most successful live attenuated vaccines is Yellow Fever virus (YFV) vaccine based on live attenuated strain of YFV, a flavivirus closely related to DENV. The vaccine strain of YFV is suitably attenuated, yet highly immunogenic. Immune response elicited by attenuated YFV provides long term protection from YFV infection. The close genomics and antigenic relatedness of YFV and other Flavivirus make it conducive to prepare chimeric viruses in which the backbone of the attenuated YFV is retained and genes for structural antigens are replaced by those of other flavivirus (6). Monath and co-workers (21) have produced a series of chimeric YFV-DV strains expressing antigens from each of the four serotypes. A tetravalent vaccine formulation produced by mixing the four monovalent vaccines in equal proportions was shown to be safe and effective in Cynomolgus monkeys (11). In the year 2000, Guirakhoo et al made the first YFV-17D dengue chimera (12). These vaccines, and later on other chimeric flavivirus were constructed by replacing the genes for YF vaccine (YFV 17D 204) pre-membrane (prM) and envelope (E) proteins, with those of heterologous flavivirus (5, 11, 21). PrM and E genes were derived either from wild-type viruses without modification (example dengue and veterinary West Nile vaccines), or from empirically derived attenuated vaccine (example JEV strain SA14-14-2 for JEV vaccine) or by introduction of specific attenuating mutations into the wild-type E by site directed mutagenesis. In contrast to neurotropic flavivirus such as TBEV and JEV (11) where residues involved in virulence had been previously identified to lie within the envelope proteins, no such residues were known for non-neurotropic viruses such as dengue. Consequently, for construction of chimeric vaccine viruses for non-neurotropic viruses, includingdengue, it was hoped that the wild type E sequences linked to the YFV 17D backbone would be suffice to mitigate the chimeric viruses. Compared to live-attenuated vaccines obtained by empirical passages, chimeric based vaccines are rationally designed, starting from cDNA to plaque-purified clonal vaccines. Further scale-up of viral vaccine production from laboratory scale to GMP manufacture requires multiple rounds of virus amplification, which have to be strictly controlled in order to avoid loss of critical attenuation properties. The seed lot system (primary or master seed lot and secondary/ working seed lot), first implemented in 1945 for the production of YFV 17D vaccine, is intended to limit the extent of derivation of a given strain, by defining and restricting the number of amplification passages. The consistency of viral vaccine properties has to be demonstrated at all steps of the full-scale production process and beyond bulk stage production for the most critical ones (by performing additional passages to further establish genetic stability). This guarantees the robustness of the manufacturing process, underlying the safety and efficacy of the product. In the YFV 17D a chimera, has been constructed for each of the four serotypes of DV (CYD serotypes). These vaccines are currently undergoing Phase I clinical trials. The main objective in chimeric vaccine is to retain the well characterized attenuation phenotype of the YF-17D backbone but incorporate dengue antigencity. The strains used in the vaccine grow well in Vero cell culture and are reported to be sufficiently attenuated and immunogenic, as evaluated in pre-clinical and clinical studies (14). The pre-clinical data has indicated that for the YF-flavivirus chimeras, the chimerization process itself is attenuating, thus augmenting the stability to the attenuation phenotype of YF-17D.
Appropriate tools and assays have been developed to measure immune responses in the pre-clinical and clinical evaluation of dengue vaccine candidates, skin dendritic cells (DCs) are amongst the first cells encountering virus after infection. DCs are the most efficient antigen presenting cells (APC) involved in the primary response to infection/ vaccination. Through usage of the four CYD stereotypes, analysis of CYDs infectivity for monocyte-derived human DCs took place(4). CYD1-4 induced DC maturation and a controlled response, accompanied by limited inflammatory cytokine production and consistent expression of anti-viral type I interferon, which showed that these serotypes have good clinical safety profile and immunogencity (7). The surrogate assay for vaccine potency of a live vaccine is in-vitro titration, assuming that protection induced by the vaccine potency of a live vaccine is linked to the infectious dose of virus injected. However, following reports of adverse reactions in individuals due to excessive dose of virus, it has become necessary to monitor the total number of particles to be injected (25). In the case of an YF vaccine, it was previously demonstrated (1) that viral infectious titration can in fact be conjoined with the total viral load estimated by RNA genome quantification and used as an index for production cohesion. For dengue vaccines based on the YFV 17D backbone, a quantitative, realtime, reverse transcriptase PCR (qRT-PCR) system was developed. One essay targeted the NS5 gene in the YFV 17D backbone, and four assays targeted the E/NS1 junction of each chimeric virus (20). The RNA copy number per infectious unit consistently ranged between 3.0 and 3.3 log10, similar to that reported for commercial YFV 17D vaccine lots (1). Furthermore, viral particles reproduced through the vaccine development pathway (cell monolayers, biogenerators, serum-free process) demonstrated by cryo-electron microscopy the typical flavivirus morphology presented in the maturation of the flavivirus. (19, 14). It is critical to address genetic stability in the course of RNA virus vaccine development as RNA viruses have the ability to recombine within and among species. In the instance of live attenuated strains obtained empirically on a number of cell substrates, the materialisation of mutants with possibly amplified or lowered virulence is inevitable across multiple passages, even after adaptation to Vero cells (17,24). In this regard, the seed lot strategy has been remarkably successful in maintaining the attenuated phenotype of YFV-17D vaccine over decades. The YFV-17D vaccine genome is remarkably stable compared with most other RNA viruses, both in vivo and in vitro (1). This high genomic stability may be attributed to the low error-rate of YFV-17D virus RNA polymerase which is also responsible for viral replication of the chimeric based vaccine strains. Lack of suitable animal model for DENV has often been cited as a reason for incomplete understanding of the mechanism of immunity. Rodent and non-human primates (NHP) models can be utilised to procure information pertaining to the attenuation and safety of flavivirus vaccines .
Comparision of Chimeric based vaccines with other vaccine candidates
Ideally a vaccine should induce both humoral and cellular immunity. Consequently, live-attenuated vaccines would be optimal. Several attenuated monovalent and tetravalent vaccine candidates have been evaluated (9, 13). Tissue culture derived vaccines were developed from viruses isolated from dengue patients and then attenuated by sequential passage in primary dog kidney (PDK) cells or primary green monkey kidney (PGMK) cells. Four serotypes of live attenuated DENV vaccine candidates were used to prepare a tetravalent formulation known as the Mahidol vaccine. Seroconversion rates for these candidates, when tested as monovalent, bivalent, or trivalent formulations, were found to be 90ââ‚¬"100% (2). Attenuated strains must be able to replicate sufficiently well in vivo to provoke an immune response (ideally against all four serotypes at the same time), but be restricted in systemic replication sufficiently to avoid inducing any of the dengue-associated symptoms. It is also necessary that live-attenuated vaccine strains be genetically stableowing to that fact that any reversion, during vaccine batch manufacture process or following administration, could possibly create adverse safety issues.It is further necessary thatthese strains be impervious to transmission by mosquitoes. This is because such transmission can lead to evolutionary change towards virulence. Transmission to mosquitoes will be unlikely if viremia is low, but mutations restricting replications in the mosquito host are also desirable. Inspite of the research into development of good live attenuated vaccine candidates, problem in developing and formulating a tetravalent DV vaccine persists (25). Striking a balance between immunogenicity and attenuation of each DV serotype and attaining uniformity of immune responses to four serotypes in a mixed tetravalent formulation is difficult with problem of reactogenecity, serotype dominance and competition. Researchers have described numerous different DENV1-4 live vaccine candidates derived from clinical isolates by sequential passage in PDK cells. Several of these, although attenuated in preclinical studies, were subsequently found to be unacceptably reactogenic in human trials. (13). Through production or introduction of mutations into the genome, DENV can be mitigated.. Passage of DENV in PDK cells has led to the accumulation of mutations associated with an attenuation phenotype and specific mutations derived by this empirical approach were identified in 2000 as contributing to the attenuation of the DENV-2 PDK-53 vaccine candidate. In a separate vaccine strategy, the DENV-4 full-length cDNA clone was used to engineer deletion mutations into the 30-UTR of DENV-4, which conferred varying levels of attenuation in rhesus monkeys compared to the wild-type parent virus. (13) The DENV-4 virus containing the D30 mutation (DEN4D 30) was evaluated in adult human volunteers and was shown to be safe, asymptomatic and immunogenic (8). The success of the DEN4D30 vaccine in humans supported a unique strategy to create vaccine candidates for the other three DENV serotypes, Since the structure of the DENV 30-UTR is well conserved among all four serotypes, it was reasoned that deletion of nucleotides analogous to the D30 mutation in each serotype would likely result in attenuation. However, introduction of the D30 mutation into DENV-2 conferred only a modest level of attenuation and introduction into DENV-3 failed to attenuate the resulting virus (3). Recombination is also a potential concern when dealing with mixed formulations of live/replicating viruses. RNA viruses can recombine within and between species. This led many researchers to develop inactivated vaccines as they have three perceived advantages over live attenuated vaccines. These vaccines cannot revert to more virulent viruses, they will not interfere with each other in a tetravalent formulation and they can be given to persons who may be immunocompromised. However, these vaccines are generally more expensive to produce, require multiple doses and do not induce the broad or long- lived immune response of live vaccines. With the development of molecular techniques, lot of research has been made in the field of sub-unit vaccines. One such candidate is the recombinant subunit protein vaccines. Recombinant subunit protein vaccines are being evaluated as alternative vaccine strategies to avoid some of the issues encountered with live attenuated vaccines. Recombinant DENV proteins can be expressed in baculovirus, yeast Escherichia coli, vaccinia virus and mammalian cell and then purified for use as non-replicating subunit vaccines. Full- length E protein, without the co- expression of prM, is targeted intracellulary and is not secreted, failing to induce neutralizing antibody. However, when prM and E are expressed together, the integrity of the neutralizing eptiopes is maintained. For this reason, the E protein co-expressed with prM has been most extensively studied in subunit protein vaccines and the baculavirus expression system has been most widely utilized for the expression of these proteins (9,13). Further research is required to develop an ideal DV vaccine candidate. Another latest technology aims to utilize the DNA as vaccine agent. It has been proposed that DNA vaccinespossessa number of advantages, compared to traditional inactivated whole virus vaccines, recombinant protein vaccines, subunit protein vaccines and, to some extent, live attenuated vaccines (13). DNA is stable for long periods of time and is resistant to extremes of temperature, overcoming cold-chain restrictions. Indeed, it is clear that the proteins produced by DNA vaccines are translated and processed within the host cell. As a consquence these proteins able to induce class-I MHC- dependent immune responses. In addition, DNA vaccines cause less reactogenecity than live vaccines. However, they are unable to induce long - lived humoral and cellular immunity, the DENV-2 neutralizing antibody titers induced by the DNA shuffle vaccines were not sufficient to protect against DENV-2 infection and there is a need to evaluate the strategies to enhance this immune response (13). Virus vectored DENV vaccines have also been studied. Recombinant poxviruses and adenoviruses expressing foreign proteins have been demonstrated to induce strong humoral and cellular responses in humans against various pathogens (9, 13). However, early studies of recombinant vaccinia viruses expressing the structural proteins of DENV-2 or DENV-4 were disappointing (74). These constructs failed to induce neutralizing antibody and failed to protect monkeys from wild- type challenge.
In conclusion, it is seen that the pathogenesis of dengue fever and the mechanism of immunity against DV is not clearly known which poses a challenge to development of ideal vaccine candidate against DF. Among all the options presently available, the YFV17D ââ‚¬"DENV1-4 chimera based vaccine candidate has the potential to provide life long immunity, induces low reactogenecity and is effective against all four serotypes of DV.