Vaccines Against Dengue and West Nile Viruses in India: The Need of the Hour

Abstract

The circulation of flaviviruses, dengue (DEN), Japanese encephalitis (JE) and West Nile (WN) viruses, and others, is generating a major concern in many countries. Both JE along with DEN have been endemic in large regions of India. WN virus infection, although circulating in southern regions for many years, in recent years, WN encephalitis patients have been demonstrated. While vaccines against JE have been developed and decrease outbreaks, in case of DEN and WN, vaccines are still in developing level, especially, it has been difficult to achieve the long-term protective immune response. The first licensed DEN vaccine, which is a live attenuated vaccine, was administered in countries where the virus is endemic, and has a potential to cause serious side effects, especially when administered to younger population as observed in the Philippines vaccination drive. In the case of WN, although the purified inactivated virion-based vaccine worked effectively as a veterinary vaccine for horses, no effective vaccine has yet been licensed for humans. The induction of CD4⁺ and CD8⁺ T cell responses is essential to complete protection by these viruses, as evidenced by responses to asymptomatic infections. Many studies have shown that neutralizing antibody (NAb) response is against surface structural proteins; CD4⁺ and CD8⁺ responses are mainly directed against nonstructural proteins rather than NAb response. New data suggest that encapsulating virus vaccines in nanoparticles (NPs) will direct antigen in cytoplasmic compartment by antigen-presenting cells, which will improve presentation to CD4⁺ and CD8⁺ T cells. Since tissue culture-derived, purified inactivated viruses are easier to manufacture and safer than developing live virus vaccines, inclusion of NP provides an attractive alternative for generating robust flaviviral vaccines that are affordable with long-lived protection.

Introduction

Flaviviruses are the most prevalent viruses, circulating all over the world, transmitted by zoonotic vectors, and causing extensive human and animal morbidity and mortality (74). Most flaviviruses are transmitted by mosquito or tick vectors. Dengue (DEN) and Zika (ZIK) viruses have a mainly mosquito-human-mosquito transmitting cycle, while others need non-human animal/bird amplifiers. To complicate matters, DEN transmission has a transovarial transfer mechanism, which contributes to the sustained presence of this virus in nature. In humans, prolonged viremia is noticed in DEN, ZIK, yellow fever (YF), West Nile (WN), and Kyasanur Forest disease viruses. Only DEN has four different serotypes causing multiple infections. It should be noted that vector control and antiviral agents have not been yet been successfully applied in the control of transmission of these viruses. This is why prophylactic vaccination is the only measure for preventing morbidity and mortality in humans (46,75).

Morbidity and Variation of WN and DEN Virus Infections

Japanese encephalitis (JE) and DEN infections, in addition to WN, form a major portion of endemic and seasonal arboviruses with a high cumulative morbidity in areas such as the Indian subcontinent. To develop newer concepts or improve current vaccines, it is necessary to understand the extent, clinical presentation of symptomatic infection, and immune response in these viruses. In India, asymptomatic infections had been reported for many years with isolations from mosquitoes and humans, especially in South India. Febrile illness in epidemic form and clinically overt encephalitis patients has been observed in Assam, North east (62), and Kerala, Southern regions of India (3,13), and recently, in West Bengal and Madhya Pradesh states (64,129).

Worldwide, WN is one of the most widely distributed arboviruses and an emerging threat. WN has been causing morbidity and mortality in humans and animals from Eastern Europe for years. The disease was introduced in 1999 in the United States causing crow and human morbidity and mortality (67), and is currently labeled as seasonal outbreaks. WN has been detected in China (17) and Brazil as well (86). Persistence of WN has been demonstrated in human subjects, requiring screening of blood donors for the presence of WN virus (17,18,80). Ratio of asymptomatic versus symptomatic infections and rate of persistence is currently not known. Virus is transmitted by Culex spp., causing overlapping infections of JE-WN in India. St. Louis encephalitis virus, which was circulating in the United States, has been replaced by WN.

In a serosurvey study in normal population, DEN in India is endemic and ranges from 76.9% in the southern region to 60.3% in the north (90). Worldwide, ∼2.5 billion people are at risk of DEN infection every year, with estimated symptomatic patients of about 390 million per year, of which 96 million manifest clinical symptoms (98). The four serotypes of DEN virus, although closely related, do not protect each other from acquiring subsequent infections. Virus transmission by the human-Aedes spp. mosquito cycle contributes to difficulty in controlling the circulation of the virus. The broad spectrum of disease in humans varies from asymptomatic to severe hemorrhagic fever, multiple organ failure, and death.

During the 1980s, DEN was thought to be a self-limiting infection, although this was disproved on encountering severe DEN outbreaks, including dengue hemorrhagic fever (DHF), mainly in Thailand (92,93). DHF was mainly observed in patients having an earlier history of infection by a different DEN serotype. World Health Organization (WHO) has developed a system of DEN diagnosis by classifying the severity of DEN (59). Usually, secondary infection with a virus with a different serotype and rapid increase in viral load effect appear to be a major parameter (59,127), along with thrombocytopenia. In recent time, various reports of central nervous system (CNS) infections, ranging from meningitis to meningoencephalitis, have been reported. In Brazil, a study of 209 patients showed 3.8% to be positive for DEN meningitis (4), and of 10,107 patients in a study in India and Sri Lanka of patients presenting with acute encephalitis, 5.2% showed DEN CNS symptoms (141). These studies press home the urgency for manufacturing the DEN vaccine.

Studies in Mice

Mice are useful in studying various immune responses to WN and DEN infections, vaccine potency, and pathology. Although not the natural route of infection, all flaviviruses can infect and cause mortality by intracerebral or intraperitoneal (IP) injection in infant mice. Adult mice are not susceptible to most of JE and DEN infection by peripheral injection such as IP, with Genotype I (G I) of WN being an exception (13) along with JE strains like Sarawak, P3. This is probably by stabilizing blood–brain barrier (BBB) by age of 21 days (10). Absence of adult challenging model is the biggest problem in studying efficacy of a candidate vaccine. To overcome this, temporarily compromising BBB by intracerebrally injecting 1% starch can render adult mice susceptible to peripheral IP administration of virus (42).

This model was used in various studies on DNA and chimeric peptide vaccines (1,30,66). Weanling mice have a permeable BBB and delays brain function. Thus peripheral administration of virus in these animals for sufficient time to study host-virus interaction, and cellular immune responses. Adoptive transfer of various T cell populations, followed by peripheral virus administration, was used as a major method to study dominant cellular immune responses against the flavivirus, JE (10,54). Currently, to study pathogenesis of DEN, ZIK, and other viruses, AG129 mice deficient in both the interferon-α/β receptor and the interferon-γ receptor are available (119). This strain, however, cannot be used to reproduce normal immune response even to inactivated protein;, AG 129 mice were used for testing recombinant attenuated DEN vaccine (109). Few studies have been carried out by testing in nonprimate models also (96). There is therefore a need to develop other animal models that will truly recapitulate the immunization and challenge by infection through peripheral route as observed in humans.

Immune Response Against Flaviviruses

The major objective for any vaccine development project is that it has to be low cost, effective in single or two doses, capable of inducing long-term memory response, and with low side effects. Ironically, asymptomatic flaviviral infections manage to achieve most of these objectives. Various studies comparing asymptomatic and severe flaviviral infections (7,47,135) have revealed that subclinical infections include all the arms of immune response against every protein expressed by the virus to overcome negative effects.

Antibody Response

Neutralizing antibody (NAb) makes up the major arm of the immune response against flaviviruses, and is used as an indication for diagnosis and protection in flavivirus infection. The IgM antibody response is detected early and is used as a diagnostic method for recent infections. However, to unequivocally confirm specificity of a recent infection, dual testing of serum samples from early and late stages of infection by IgM capture enzyme-linked immunosorbent assay (ELISA) as well as in vitro NAb test is recommended (3). Only E protein is responsible for NAb and not nonstructural (NS) NS1 and NS3. As it will be shown below that, rather than E protein, NS1 and NS3 generate T cell responses. Analysis of amino acid sequences among different proteins of JE, WN, and DEN, using in silico method, demonstrates global similarity in score of NS3 protein between 83 and 64, envelope (E) protein between 82 and 44, and NS1 between 80 and 54.

While the high cross-reactivity in antibody-specific responses to the E protein of ZIK and DEN makes it challenging to perform differential clinical diagnosis, the NS1 protein is highly specific and can provide a better option for differentiating between these two flaviviral infections for current infection (40). In India, due to co-circulation of JE, WN, and DEN, reactivation of memory B cells may occur, which may be cross-reactive in nature (150). To complicate matters, it is also difficult to differentiate between IgM responses elicited by the attenuated JE vaccine (SA 14 14–2) and recent infection by related flavivirus. The NAb response in WN also helps in resolution of infection (5), as observed in mice where passive transfer of protective WN antibody in Rag^−/− mice was able to limit acute disease. However, this protection did not persist, causing severe WN disease and death, suggesting a role for T cells in clearing virus (34).

DEN has four serotypes, co-circulating in a given geographical location. Although these are closely related with only minor difference in genetic and proteins, NAb response against one serotype does not protect against another (46,75). Antibody-dependent enhancement (ADE) of infection is an immunopathological phenomenon, whereby monocytes or macrophages take up infectious immune complexes and are infected at a higher rate (49,50), with the potential to result in DHF (92,93,114). This is mostly specific to DEN infection, through Fcγ receptor-mediated uptake of immune complexes. Two immunodominant epitopes, one to the precursor membrane (PreM) protein and another to the fusion loop epitope on E protein, are recognized by cross-reactive antibodies that are not only poorly neutralizing but can also promote increased viral replication and disease severity (123). Heterotypic secondary infection with DEN increases the likelihood of severe disease, although the majority of secondary infections are mild or asymptomatic (106).

Individuals living in endemic regions, who did not suffer from severe disease, demonstrated binding antibodies in >80% individuals (47). Long-term travelers from the United States to South Asia showed both polyclonal as well as cross-reactive NAb responses, suggesting that, broadly NAb may emerge depending on the number of previous exposures to different DEN serotypes (100). A study carried out in Nicaragua on secondary DEN 2-infected subjects suggests that increased DEN disease severity is associated with a higher decay rate of antibody avidity at later time points postillness (70). In DEN-3 infections, primary and secondary DEN-2 infection demonstrated that anti-DEN 3 was low in primary infection and increased during the convalescent phase, and decreased further. It appears that in DEN fever (DF), the patient's IgM can control infection and this is aided by an earlier infection (106), highlighting the need for a better understanding of NAb in polyclonal human sera (134). The recent phase 2b and 3 trials on tetravalent DEN vaccine (EN-YF) reported only a moderate efficacy, despite the presence of NAbs. Furthermore, DEN recombination vaccine using DEN 1,2,4 background with DEN 2 PreM and E has been in phase III level with excellent response.(8,133).

The role of NS proteins in DEN infection is also important (20). The NS1 protein contributes to vascular leakage, coagulopathy, and thrombocytopenia. Some antibodies to NS1 may cross-react with endothelial cells. Appropriate management of patients has demonstrated that apoptosis of endothelial cells induced by DEN infection or anti-NS1 antibodies is not sufficient to support the clinical outcome (6,50). In addition, NS1 antibody titers in DF or in healthy individuals with past no-severe DEN infection had similar antibody repertoires (55).

T Cell Response in Flaviviruses

The protective role of T cells during viral infection is well established. Generally, CD8⁺ T cells can control viral infection through several mechanisms, including direct cytotoxicity, and production of proinflammatory cytokines such as IFN-γ and TNF-α. Similarly, CD4⁺ T cells are thought to control viral infection through multiple mechanisms, including enhancement of B and CD8⁺ T cell responses, production of inflammatory and anti-viral cytokines, cytotoxicity, and promotion of memory responses. Engagement of both CD4⁺ and CD8⁺ T cell responses is necessary for protection to flavivirus infection. Dominant T cell epitopes are mainly located in the NS region of the flavivirus polyprotein. E protein, mainly responsible for NAbs, has fewer T cell epitopes.

In JE, memory phenotyping of T cells from recovered patients demonstrated mostly CD4⁺ T cells against both structural proteins and NS1 (65). T cell receptor (TCR)α/β knockout mice supported JE infection, developed minimal IgG responses, and were protected by adoptive cell transfer of CD8⁺ T cells from wild-type mice (54). We have also demonstrated that Th2 CD4⁺ T cells against NS proteins are protective (10). In asymptomatic individuals, memory cells were mostly IFNγ-producing CD8⁺ T cells against NS proteins (135), while in the case of WN, it appears that, despite homology with JE, protective T cell responses appear to be mainly CD8⁺ T cells. Detailed screening of overlapping WN-specific peptides in PBMCs revealed that CD8⁺ T cell peptides for M, E, NS3 NS4b and NS5, and NS4b proteins dominated (68,126). Similar observations for memory responses for CD8⁺ T cell epitopes were detected (29,60,99). Nonetheless, an exuberant CD8⁺ T cell-mediated response can lead to injury and death of infected or bystander neurons in the case of CNS infection (21,33).

Conversely, the lack of a functional CD4⁺ and CD8⁺ T cell response results in inefficient clearance of WN infection from neurons (140). In mice lacking CD4⁺ T cells or class II MHC, enhanced disease and mortality demonstrated contribution of CD4⁺ T cells (14). Furthermore, after identifying CD4⁺ T cell epitopes, peptide vaccination of immunocompetent mice resulted in increased survival (33). In addition, in WN infection, elevated viremia led to a greater dissemination to the CNS in γδ T cell-deficient mice, revealing a possible role for these nonclassical T cells (145,146). In both human and primate models, γδ T cells may not be directly involved in memory response to WN infection. TCRδ ^−/− mice displayed a numeric and functional reduction in both CD4⁺ and CD8⁺ memory T cell responses (143,144).

The role of T cells in DEN infection and subsequent disease manifestations is not fully understood. Original antigenic sin refers to the propensity of the body's immune system to preferentially utilize immunological memory based on a previous infection when a second slightly different version of that foreign entity is encountered, as is frequently seen with secondary infections of a different DEN serotype. Depending of the first viral exposure, the secondary immune response can result in ADE or conversely, could induce anergy. Both these responses can potentially trigger a loss of pathogen control and induce aberrant clinical consequences. Profound T cell activation and death may contribute to the systemic disturbances leading to DHF, leading to higher viral loads and increased immunopathology due to cytokine storms (89,142). In secondary infections against a different serotype, low-affinity T cell response may also lead to immunopathology (113). A detailed analysis of the anti-DEN E protein domain 3 response in a cohort of patients suffering either primary or secondary DEN infections demonstrated dramatic evidence of original antigenic sin in secondary infections, both in terms of binding and enhancement activity (86,146).

A comprehensive overview of CD4⁺ and CD8⁺ T cell epitope reactivities against the DEN 2 proteome in adult patients experiencing secondary DEN infection demonstrated that CD4⁺ T cell response is mainly against structural and NS1 proteins, and is responsible for the development of high-avidity antibodies (53,111,112). These results also indicate that CD4⁺ T cells help in the development of high avidity antibody in subsequent infection (112). Similarly, CD4⁺ and CD8⁺ T cells are persistent in the skin of infected patients (113). Data generated on CD8⁺ T cells indicated that in secondary acute stage DEN infection high level of cross reactivity of CD8⁺ T cell is detected, whereas during convalescence, patients demonstrated higher avidity serotype-specific clones (31). Using an array of DEN peptides from entire polyprotein, DEN IgM-positive patients from India and Thailand in acute stage of infection expressed markers indicative of antigen-driven CD8⁺ T cell proliferation, tissue homing, and cytotoxic effector functions, most commonly against the NS3 protein (19).

Similarly, in asymptomatic individuals from different regions, DEN infection elicited a broad antiviral T cell response with NS3, NS4b, and NS5 being the main targets for CD8⁺ T cells (111,112,147). The majority of CD8⁺ T cell IFN-γ responses were associated with an effector memory phenotype. Infected cells did not co-express other inhibitory receptors and were able to proliferate in response to DEN-specific stimulation (28). Similarly, analyzing the TCR profiles of DEN-specific CD8⁺ and CD4⁺ cells gives the information about different levels of response in population. It was observed that DEN-specific CD8⁺ T cell mainly consisted of effector memory subsets (131). Studies on CD4⁺ TCR showed that HLA-DO, DQ, and DRB3/4/5 loci mediate had lower magnitude compared to HLA-DRB1 (45). In CD4⁺ T cells, response in vaccinees was similar in magnitude and broader to those after natural infection (2).

Regulatory T Cell Response

Since the discovery of regulatory T cells (Tregs), it has become clear that this population is responsible for maintaining T cell homeostasis in both infection and autoimmune disease. Tregs are a subset of CD4⁺ T cells, which can regulate effector CD4⁺ and CD8⁺ T cells, and have been shown to play an important role in a variety of viral infections (61). In humans infected with WN, higher Treg cell frequencies were found in PBMC. Both preclinical subjects and patients exhibiting clinical symptoms had lower Treg frequencies compared to asymptomatic individuals (44,68,69). These observations suggest that Treg cells may play a role in limiting WN disease, perhaps by limiting pathogenic aspects of the immune response, kinetics of infection, and migration of effector cells (91). Similarly, in two studies with varying clinical symptoms, significantly higher frequencies of Tregs were observed in mild cases, corresponding positively with platelet counts and negatively with interleukin (IL)-6 and IL-8 levels (81,132). In addition, a positive correlation with platelet counts and beneficial outcome was observed (132). As mentioned above, Tregs help in higher IgG avidity in convalescence (31).

Vaccine Development

At present, there are a few standard and some recently developed methods to generate vaccine development. Cell culture-based purified inactivated virions, and attenuated virus strains are used all over the world. This can include expressing flavivirus proteins in vectors such as vaccinia or adenovirus. Historically, NAb has been regarded as the major measure of protection in all vaccine trials (140). However, as detailed above, other arms of the immune response are equally as important for effective and long-lived protection. Memory cells are not only necessary for protection from secondary responses but it has also shown that sequential immunizations for flaviviruses sharing CD4⁺ T cell epitope can promote protection during a subsequent heterologous infection (72,116).

Advantages of the live attenuated vaccine are that it can elicit CD4⁺ and CD8⁺ T cells as well as an antibody response. Another approach is a cDNA vaccine, which is based on a plasmid expression vector. Inserting a cell-specific promoter can restrict expression to antigen-presenting cells, while de-targeting expression from other cell types (1). As an alternative to the inactivated virion-based vaccine, expressing structural antigens by mammalian or yeast systems have been tested as vaccine candidates. Encompassing these antigens in virus-like particles (VLP) has proven efficacious for the hepatitis B virus hepatitis B surface antigen (HBsAg). In addition, to generate highly specific portions of protective region of a protein, B and T cell epitopes as immunogens have been extensively studied (66). Preparation of chimeric T helper and B cell peptides has also been used as immunogen (30). CD4⁺ T cells help in generation of both B cell and cytotoxic T lymphocytes (CTL) response (15). In silico methods to predict peptides for antibody binding or MHC I or MHC II are available.

Vaccines against encephalitis flaviviruses

Few of flaviviruses cause meningoencephalitis infections with high mortality. As mentioned above, JE is highly rampant in South East Asia, while WN neurotrophic infections are circulating in Eastern Europe and since 1990s has caused encephalitis in USA. Cumulative 24,714 neurological disease cases and 2,314 deaths in the USA since 1999 (58). Many anti-JE vaccines are successfully controlling over the years. Prophylactic vaccines for JE, which are currently used, are attenuated JE SA 14-14-2 and Vero cell-based inactivated purified vaccines (26,52,121,135). An independent study in India on the efficacy of a single dose of attenuated JE vaccine has shown that overall efficacy was 72.2% (128,136,141), or 62.3%. In India, SA 14-14-2 has been used in endemic regions since 2006. A JE-YF recombinant (ChimeriVax/Imojev), using YF 17D vaccine as a vector, has been approved and shown to generate sustained NAb responses (16,22). In addition, a VLP using G I E protein was able to protect swine against G III infection (35).

In case of WN, currently, four licensed veterinary vaccines are available for horses; no vaccine has been approved for human use (48,58). Comparison of inactivated and recombinant vaccines has also been carried in animals and demonstrated similar immune response (88). Newer methodology of inactivation by hydrogen peroxide-based agents has been undertaken (108). However, in this, including cupric ions complexed with the antiviral compound, methisazone might cause concerns in future (108). Most vaccination strategies against viruses focus on antibody neutralization. However, given the importance of CD8⁺ T cells in clearing viral reservoirs in WN infection, an effective vaccine would have to target both arms of the immune response. A JE-inactivated vaccine JE-ADVAX was able to provide heterologous protection against WN infection, especially when combined with existing inactivated WN vaccine for horses (102).

Recombinant WN E protein formulated with the particulate, saponin-based adjuvant Matrix-M™ induced strong cellular recall responses in splenocytes (82). Similarly, monkeys inoculated with E protein produced in Drosophila melanogaster mixed with an adjuvant (GPI-0100) (73) or with PreM-E protein (154) responded by lymphoproliferation, cytokine production, or NAb, respectively. E protein-based phase I trial was conducted in limited individuals, resulting in a NAb response (149). The company has now acquired rights for technology from Merck Inc. for protein-based vaccine (51).

WN DNA vaccine formulated with PreM-E protein gene did not show a significant E protein-specific humoral response. However, protein boosting of DNA-primed animals resulted in a marked increase in total NAb titer (29). Using recombinant WN-vectored vaccine based on a fish Novirhabdovirus, the viral hemorrhagic septicemia virus induced protected 40–50% of mice against WN lethal challenge (94). RepliVAX WN is a novel WN vaccine based on single-cycle flavivirus particles (137). This as well as YF WN PreM-E chimeric vaccine have shown promise and demonstrated T cell differentiation from an effector phenotype to a long-lived memory phenotype (27,124). WN-YF chimeric live virus vaccine is being studied in phase I level (43). Using neuraminidase protein-based D III domain of WN has shown NAb immune response and protection (85). It is essential to remember that CD4 T cell response is essential to develop a robust CD8⁺ T cell response. Various studies clearly demonstrate a role for CD8⁺ T cells in vaccine-induced protection from WN and suggest that generation of a robust T cell response should be an important component of future vaccines.

DEN vaccines

In the DEN scenario, developing “the successful vaccine” still appears to be a distant aim (41). The enormity of DEN infection, severity, and four serotypes, along with other flaviviruses circulating in the same region largely complicate matters. Based on information seen in other virus vaccines, it has become clear that monomeric E protein does not work on its own and needs an adjuvant (24). Preclinical studies have explored DNA plasmid expressing different proteins singly or in combination (25,122). A truncated DEN E protein (DEN-80E) for all four serotypes was used in a three-dose schedule in a phase 1 placebo-controlled/dose escalation (83). Using an adenovirus prime/plasmid boost strategy vaccine for immunizing has also shown promise (63).

PreM-E and E protein VLPs using a Pichia pastoris system (79,103,138) as well as a recent approach utilizing C-PreM-E VLP, together with a modified complex of the NS2B/NS3 protease (11), have been developed. Interestingly, generating VLPs at 31°C, which maintains the nativity of the VLP, showed a better response. Major studies have worked on preparation of DIII domains of DEN 1–4 on the HBsAg background (109). This work presented defined data on protection, protection in mice model, as well as immunity in monkeys.

Two recombinant attenuated DEN vaccines have been developed. The first licensed DEN vaccine, Chimeric Yellow Fever Derived-Tetravalent Dengue Vaccine (CYD-TDV) (Dengvaxia^®), has received regulatory approval in about 20 countries and is implemented in two countries. However, this vaccine has some limitations. In monkey experiments, differential results for different serotypes were observed (139). A WHO-directed study over the years after conducting phase I to III trials in many locations and analysis of data concluded that Dengvaxia has the potential to reduce the burden of DEN disease in areas of moderate to high DEN endemicity. However, the potential risks of vaccination in areas with limited exposure to DEN as well as the local costs and benefits of routine vaccination are important considerations for the inclusion of Dengvaxia into existing immunization programs (37,38).

Further analyzing Philippines DEN vaccination program, it was observed that about 70% protection was seen, while breakthrough was also seen. Substantial number of severe DEN cases and, among those that do occur, the majority are likely to be breakthrough disease in seropositive vaccinees and a minority attributable to the excess risk of enhanced disease in seronegative vaccinees (76,148). Thus, the Strategic Advisory Group of Experts (SAGE) found that the DEN 1-4-YF vaccine developed substantial seroconversion and protection in earlier seropositive individuals. However, there was an excess risk of severe DEN in seronegative vaccinees (148.)

Another attenuated vaccine DENVax (R) or TDV is a tetravalent recombinant live attenuated dengue vaccine (LATV) based on a common virus backbone (32): a molecularly cloned, attenuated DEN serotype-2 strain; this vaccine has been developed by the Centers for Disease Control and Prevention (CDC) and Inviragen (now licensed to Takeda). Another LATV is TV003/TV005 (R), developed by NIAID (95,105). Earlier studies on non-flavivirus-exposed individuals demonstrated adverse effects (AEs) in the form of a rash in 64% vaccinees (29). In a further randomized, multicenter phase 1b study conducted in the United States, the safety and immunogenicity of TDV were evaluated (115). A large study was carried out in Columbia for safety and immunogenicity of TDV. Adult individuals enrolled were negative for antibodies to all DEN virus serotypes and to antibodies for YFV, WNC, HBV, HCV, and HIV, and given two formulations containing different ratios. After administration of the first dose, levels of DENVax seroconversion ranged between 24% and 100% for each serotype. After two doses, the highest rates of seroconversion were consistently noted for serotypes 1 and 3, followed by serotype 2, and then serotype 4. Viremia was noticed in many individuals also (97).

Surprisingly, a classical method of purified inactivated virion production using a tetravalent vaccine (DPIV) was also undertaken. Initial studies in non-human primates demonstrated that two doses of DPIV administered 4 weeks apart protected for 8–10 months, although viremia was observed following challenge with live, near wild-type DEN-1 and DEN-2 (36). This study was extended to a placebo-controlled, randomized, observer-blind phase 1 trial. No vaccine-related severe AEs were observed through 12 months, and geometric mean antibody titers peaked on day 56. In the nine subjects where boosting was evaluated, a strong anamnestic response was observed (118).

Nanoparticles as Carriers for Inactivated Protein-Based Vaccine

A recent review by Rey FA et al. (110) showed that the quasi-structure of E protein on a virion can cause a high level of cross-reactivity due to exposure of the fusion loop in acidic pH. Furthermore, in a live virus vaccine, all the forms of immature and mature virions are available for mounting an immune response. This might be the reason for getting a high cross-reactive response in live attenuated vaccine. Formalized virion or protein at proper pH might improve its specificity. Recent studies are now directed at developing an appropriate adjuvant or delivery methods to specific cells such as antigen presenting cells (APC), and compartments. Nanoparticles (NPs) are therefore an attractive delivery tool (12,153). Many NPs are clinically approved and have been used in peptide-DNA delivery approaches such as for influenza viruses. Nps can be helpful to deliver protein in cytoplasmic compartment to generate MHC I-based immune response (125).

During the previous decades, hundreds of adjuvants and adjuvant formulations have been proposed as immunostimulants for vaccines or for cancer, but very few have been used in human vaccines due to toxicity concerns (9,23,39,77). Inorganic NPs and silica-based NPs are nonbiodegradable, while the use of metal NPs has been strictly restricted to gold and silver (151). VLP-based vaccines have been the first NP class to reach the market (117). Polymers such as synthetic polymers-poly (D,L-lactic-co-glycolic acid), polyethylene glycol or polyester biobeads, and natural polymers based on polysaccharides such as alginate, inulin or chitosan, and liposomes are in active research. Recently, many studies are also focused on chitosan as a substrate, alone or in combination with other reagents. WHO and the Food and Drug Administration (FDA) have approved many of NP formulations for human use (120).

NP vaccines can potentiate immune responses by site-specific drainage to lymph nodes with a fine control over size and co-delivery of antigens, generating potent T cell responses (71,107). Chitosan shows particularly high biocompatibility and fairly low cytotoxicity (84). However, as it is insoluble at physiological pH, lacks charge, and shows poor transfection, alternatives such as amphiphilic chitosan linked with low molecular weight polyethylenimine (78) and poly-ɛ-caprolactone have been tested and used for HBsAg and tetanus toxoid (56,57,102,104,152). Lipid NPs have also been used for HBsAg to generate CTL response (130).

Probable Future Line of Studies Against WN and DEN Vaccines in India

It is important to initiate development of vaccine against DEN and WN, in addition to improve current inactivated JE vaccine using combined concepts mentioned above. Combining existing knowledge collected from different studies, mainly on asymptomatic immune response rather than severe complications, can help in developing more effective vaccines. T cell responses are especially important for viruses that generate high viremia. Both DEN and WN viruses have a major viremic phase, indicating the necessity of an MHC I-specific response. Approval of attenuated live vaccine needs a lengthy clearance in each country, which might delay their public health use. Also, cost of production of four DEN serotypes in one vaccine might be the cause ratio of cost and efficacy concept. Classic methods of using inactivated vaccine are still valid and the method is well documented. It is possible that expressing defined proteins, including inactivated virion into the cytoplasmic region of APC, would work. Combining this with purified inactivated native NS1 and NS3 for T cell response can also be initiated. Finally, the problem of generating CTL response by protein-based vaccine can be answered by encapsulating in NPs. Using the current approved ion exchange method of virion purification can be extended to purify NS1 and NS3 from infected cell lysates. Encapsulating these in NPs is possible as an alternative approach for developing anti-DEN and anti-WN vaccines.

Footnotes

Acknowledgments

Author is grateful to Dr. M. Biswas for improving the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

Author is supported by Indian Council of Medical, New Delhi, DHR, Ministry of Health, Government of India, as Emeritus Research, at National Institute of Virology, Pune, India.

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