The global burden caused by dengue virus (DENV) has dramatically increased over the decades [1]. It is the most common vector borne virus affecting many tropical and subtropical nations with Asia bearing approximately 70% of the burden [2]. In 2012, it was noted that dengue affected more than 125 countries worldwide [3]. According to WHO, there was a colossal rise in the number of cases, from roughly 500,000 in 2000 to over 4.2 million in 2019. Likewise, the death rate escalated from 960 in 2000 to 4032 in 2015 [4]. The Global Burden Disease Study 2017 noted that dengue was responsible for 2,922,630 disability-adjusted-life-years (DALYs) globally. This shows a steep rise of 107.9% since 1990 [1]. Furthermore, the gross global economic burden of dengue in 2013 was US$ 8.9 billion inclusive of hospitalizations, fatal and non-fatal cases [5]. Nonetheless, the number of recorded cases and the financial burden differ from the true number as cases are predominantly asymptomatic and go undiagnosed [4]. Furthermore, it was noted that countries of the low-middle socio-demographic status were more affected. This can be explained by urbanization which leads to a heightened number of susceptible human hosts [1]. Overall, dengue is considered to be a neglected tropical disease (NTD) as well as an infectious disease of poverty (IDP) [6].

Infected travellers have resulted in the transmission of dengue to non-endemic areas; additionally, dengue has replaced malaria as the most common cause of fever in returning travellers [7]. In recent years, dengue has spread to novel regions such as Europe. In 2010, the first cases were reported in France and Croatia and the first major outbreak was reported in 2012 in Portugal with an incidence of over 2000 cases [8-10]. 

Dengue fever is an arthropod borne disease due to a virus originating from the Flaviviridae family. It is now known that the dengue virus has four related serotypes - DENV 1-4 [11,4]. It is a single stranded RNA virus with three structural proteins (capsid (C), membrane (prM) and envelope (E)) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) [12]. The main mode of transmission is through the female species of Aedes aegypti mosquitoes [4]. However, another mode of transmission is from human to mosquitoes, when an uninfected mosquito feeds on a symptomatic, presymptomatic or asymptomatic individual [4,13]. It was further recorded that pre-symptomatic or asymptomatic individuals had a greater transmission potential. This is because, mosquitoes would be exposed to a higher viral load compared to when exposed to symptomatic participants [13].

Dengue is a severe flu-like illness, which rarely can be lethal. It affects all ages and is categorised as dengue with/without warning signs and severe dengue. After an incubation period of 4-10 days, the illness lasts from 2 days to a week. The main feature of dengue is a high fever accompanied by severe headaches, muscle or joint aches, nausea, vomiting and a rash. Severe dengue on the other hand, can be fatal due to increased vascular permeability leading to fluid accumulation and organ damage. At this stage, careful monitoring is required to avoid morbidity and mortality [4].

Upon infection with one serotype, lifelong protection against the serotype is ensured and partial immunity is also provided in the short-term to the other serotypes. However, exposure to another serotype in the future places the individual at an increased risk of developing severe dengue [4,11]. This is attributed to a phenomenon known as antibody-dependent enhancement (ADE) [14]. 

ADE occurs when pre-existing antibodies bind to virions to form a virus-immune complex. Instead of neutralizing the novel heterologous DENV it facilitates the entrance into immune cells through the Fcγ receptors leading to an increase in viral replication and viremia [14-15]. Additionally, ADE enhances the production of proinflammatory cytokines such as TNF-α and IL-6, increasing vascular permeability therefore leading severe disease [14].

Since there is no remedy for dengue, the most logical method to its reduction is preventative care. One of the few modes of prevention is the creation of a vaccine which is able to counter all the four serotypes of dengue virus [16]. This vaccine best be safe and be able to provide protection in the long term [17]. A vaccine would be able to prevent unpredictable breakouts which would otherwise overwhelm already fragile healthcare systems in developing countries, where dengue is most prevalent [18]. 

Vaccines

Over the years there have been numerous DENV vaccine candidates. These include live attenuated viruses, inactivated vaccines, subunit vaccines, DNA vaccines, viral vectored and virus-like-particles (VLPs) [17,19]. These vaccines target structural proteins of DENV, such as E (envelope) and prM (pre-membrane), to cause an immune response in the vaccinated individual [20]. The most developed vaccines are live attenuated and the inactivated whole virus vaccines. The remaining candidates are in the preclinical stages and require long-term monitoring to obtain knowledge about efficacy and safety profiles before these vaccines can be licensed [21]. 

The existence of effective vaccines against other flaviviruses like Yellow Fever virus (YFV) and Japanese Encephalitis virus (JEV) could provide insight to the creation of an effective vaccine for DENV. However, with DENV there are a few challenges such as the need for tetravalent response which are not obstacles faced by other viruses of the Flaviviridae family [20]. This is important as to avoid side effects like ADE as mentioned above.

Unfortunately, vaccines take a considerable duration to be created and the only licenced vaccine has become the subject of controversy [18]. The importance of this review is to provide a summary and insight about the currently available dengue vaccine candidates.

Live Attenuated Vaccines (LAV)

The first ever live attenuated dengue vaccine formulation was made in the 1940s by Albert Sabin who used serial passage in suckling mouse brains to weaken the virus strain. However, this method was rejected due to safety issues [22]. Since then, technology has come a long way and currently the most established vaccines available are LAV. The three most advanced vaccine candidates are LAV, differing in terms of the type of backbone used and the extent of chimerization [18]. This is inclusive of the only available licenced vaccine, CYD-TDV [17]. Proof of tolerability, immunogenicity and protection seen in Phase II trials show great promise for the unlicensed candidates [19]. 

Existing methods to weaken the DENV, consist of construction of chimeric viruses, targeted mutagenesis and serial passage in cell lines. A chimeric virus can be produced by replacing a wild type DENV with an attenuated form of another flavivirus (eg. YFV) or an intertypic chimera where it is replaced by an attenuated form of DENV itself [22]. In tissue culture based LAV, a major disadvantage is suboptimal immunogenicity due to the collection of mutations during the serial passage [22]. 

CYD-TDV (Dengvaxia)

CYD-TDV (Dengvaxia), developed by Sanofi Pasteur, is the only currently existing licenced vaccine approved by the FDA in 2015 [19,21,23]. The vaccine requires 3 doses to be given subcutaneously at 6 month intervals [16]. While it was licensed in Mexico and 20 other dengue endemic countries, it was only assimilated into the health programs of Philippines and Brazil [18].

Built on a YFV non-structural protein backbone (YF17D), CYD-TDV is a live attenuated, chimeric, tetravalent vaccine [17,19,20,24]. In dengue infection, neutralising antibodies form against prM and E proteins of the DENV. By replacing the prM and E proteins of the YFV with that of the four DENV serotypes, it is expected to facilitate an immune response [20,24]. The four strains (CYD 1-4) were combined to form a tetravalent formulation and they proved to have a good safety profile and be of high genetic and phenotypic stability invitro [22]. During pre-clinical assessment, seroconversion and protection against all serotypes was noted post vaccination in NHP (non-human primates). Pre-clinical assessment depicted that seroconversion and protection against all four serotypes occurred post vaccination in NHP.

In phase I trials, safety and immunogenicity were measured in humans by administering monovalent formulations. It was noted that prior immunity to YFV enhanced the longevity of cross-neutralising antibodies towards all DENV serotypes. Once safety was established, tetravalent formulations were administered in seronegative individuals. Following three doses, seroconversion was observed. Although the first dose was associated with DENV4 dominant antibody response, it did not cause ADE with subsequent doses [19,22]. 

Phase I and II trials depicted that the vaccination was safe and effective as it induced antibodies against all four serotypes post three doses, with an enhanced response in primed individuals [20,25]. The vaccine induces immunity against YFV proteins and dengue E protein which causes a skew towards IFN-G and not TNF-a hence depicting a good safety profile. Moreover, Phase II trials conducted on 9–16-year-olds in Latin America showed promising results after three doses of the CYD-TDV vaccine. Seropositivity ranged from 93.4% to 100% with higher antibody titres in flavivirus-primed individuals. Post vaccination, confirmed cases of DENV of all serotypes plummeted for the experimental group [22]. 

Nonetheless, in a phase II trial in school children in Thailand, the efficacy was only a mere 30.2% varying across the serotypes after three doses. The first dose was associated with efficacy ranging from 61-90% for DENV1,3,4, and only 3.5% for DENV2 which did not improve with subsequent doses. Additionally, cases of dengue occurred in vaccinated participants. This outcome was attributed to the DENV2 genotype residing in Thailand having differing from the DENV2 in the vaccination. Moreover, although plaque reducing neutralising test (PRNT) showed reasonable immunogenicity, it is determined using Fcϒ-receptor negative cells which is not an accurate depiction of efficacy as cells targeted by DENV typically possess these receptors [22,25]. Alternatively, other experiments also showed that the CYD-TDV vaccine induced a DENV-4 predominant response in dengue-naïve participants, although this differed in dengue-primed participants [25-26].

According to Phase II and III clinical trials, the protection rate only proved to be mediocre [17]. In two Phase III trials focusing on children and adolescents between the ages of 2-16 years in Asia (CYD14 trial) and Central and South America (CYD15 trial), the overall efficacy lied between a meagre 56.5-60.8%. The results further confirmed that in both studies the efficacy against DENV2 wasn’t up to par with the other serotypes. Both studies showed that prior seropositivity enhanced efficacy, while CYD14 portrayed that efficacy also improved with age and it was assumed that age reflected prior DENV exposure. 

Satisfactory safety profiles, were displayed in both trials. However, during the four-year long-term follow-up (LTFU) phase, the CYD14 trial showed an increased risk of hospitalization in the 2-5 age group within the first year of vaccination [19,25]. This increase in hospitalization was linked to ADE and in-vitro experiments were carried out to investigate if ADE was caused by lower efficacy against some serotypes. However, the results portrayed that the antibodies induced against all serotypes by the vaccine were similar [25]. This may be hypothesised as the amount of antibodies needed for protection may differ for each serotype which is yet to be determined through longer trials with larger populations [22,25]. Moreover, CYD-TDV carries prM and E proteins which are known to promote ADE [26]. Nonetheless, there were no safety issues with participants≥9 years, rather a satisfactory safety profile up to 25 months post first dose was noted [25].

More recently, LTFU studies have revealed that dengue naïve populations, regardless of age, have suffered an increased risk of sever disease and hospitalization [27]. Subsequently, dengue vaccination programs in the Philippines was suspended [17]. Unfortunately, although there was no formal proof, it was noted that 14 children passed away and a statistically significant number were hospitalized for severe dengue [28]. 

Since then, WHO recommends that Dengvaxia only be given to seropositive individuals therefore a pre-vaccination screening should be performed prior to vaccination [18]. In conclusion, the FDA approved Dengvaxia for the use for seropositive individuals between the ages of 9-45 years due to insufficient evidence of efficacy in the older and due to high risk of hospitalization in those younger [29].

TAK003

Having reached Phase III trials, TAK003 is another potential DENV vaccine. Like CYD-TDV, it is a tetravalent chimeric LAV formulation established by Takeda (Inviragen) [17,24]. It was inspired by the vaccine, DENVax, developed by Mahidol University in Thailand which was abandoned due to an increase in adverse events and as the tetravalent formulation did not deliver immunity towards all serotypes [17,22]. 

The backbone of TAK003 is an attenuated strain of DENV2 (PDK-53) which holds the prM/E regions of DEN1-4 serotypes [17,19,20]. The wild type DENV2 strain was attenuated by 53 serial passages in PDK (primary dog kidney) cells [22]. The presence of NS proteins due to the DENV2 backbone, differentiates it from the CYD-TDV [18]. It also enables the induction of cross-reactive T-cell response obligatory for protection [18]. Although not reported in recent studies, in an former trial, it was noted that in seronegative individuals, the vaccine provoked CD8+ T-cells targeting NS proteins [17].

Phase I and II trials of TAK003 proved to be effective and safe among all age groups and did not differ based on serostatus of the vaccinees [22,30,31]. The vaccine was able to elicit not only neutralizing antibodies against all serotypes, but also induce the humoral and cellular responses [24]. Furthermore, a lyophilized formulation of TAK003 allows stable storage at 2-8⁰C. 

A phase 2 study with 1002 dengue seronegative participants aged between 18-49 years, showed that seropositivity was achieved towards all serotypes [18]. The tetravalent vaccine induced specific memory B-cells (MBCs) for all serotypes unlike in a natural DENV infection. This was supported by a multi-colour FluoroSpot (MCF) assay, which allows differentiation between serotype specific and cross-reactive MBCs in polyclonal mixture [18]. Furthermore, other phase 2 trials showed that a booster dose or a two dose regime was essential to obtain the immunogenicity required for seronegative individuals [18].

According to Phase III trials done in Asia and Latin America, TAK003 provided 73.3% protection regardless of baseline serostatus and was effective 17 months after the second dose. Nonetheless, like CYD-TDV, TAK003 showed varied levels of protection according to serotype with the lowest rate against DENV3 and DENV4 [17,32]. Overall, TAK003 does not appear to be as affected by an individual’s serostatus as CYD-TDV, nonetheless, it is important to have a balanced protection for maximum adequacy [18]. 

TV003/TV005

The National Institute of Allergy and Infectious Disease (NIAID) of the National Institute of Health (NIH) has also established a tetravalent LAV [17,24,33]. Tetravax (TV003/TV005) was created by attenuating the DENV through deletions in the 3’ untranslated region (UTR) and chimerization of the prM/E gene. The tetravalent formulation is a concoction of rDEN1Δ30 and rDEN4Δ30 (DENV1 and DENV4 with deletions in the 3’UTR), rDEN3Δ30/31 (DENV3 with an extra 3’UTR deletion) and rDEN2/4Δ30 (DENV2 prM/E gene replaced with the same gene in rDEN4Δ30) [17,22]. TV003 formulation consisted of these components in 1:1:1:1 ratio whereas the TV005 formulation has a 10 times higher amount of rDEN2/4Δ30 to breech the gap in immunogenicity [17,24].

Early trials of TV003 showed vaccine safety and efficacy when two doses were given to seropositive participants 6 months apart. Following which, a two-dose schedule of TV003 and TV005 were given to seronegative participants. It was noted that the TV005 was better at producing a tetravalent response and the second dose only produced a less than average booster effect [17,34].

Clinical trials held in Brazil are yet to confirm the safety and immunogenicity of the TV003 vaccine. A Phase II trial experimented a lyophilized formulation in a placebo-controlled setting involving 48 healthy participants. Each of these individuals were given 2 doses 12 months apart and results depicted that a single dose was acceptable in terms of safety and efficacy [18]. Subsequently a phase III trial with 17,000 participants between ages of 2-59 is currently investigating the efficacy of a single subcutaneous dose [17]. This is in contrast to both CYD-TDV and TAK003, where both require more than one dose of vaccine for optimum efficacy [18].

TDEN F17/F19

Another likely tetravalent LAV candidate is the one developed by Walter Reed Army Institute of Research (WRAIR) with GlaxoSmithKline (GSK). The TDEN F17/F19 vaccine formulations, F17 and F19, were created through serial passages in PDK cells and fetal rhesus lung (FrhL) cells [17,24]. Out of the many formulations created, F17 showed the most promising results in flavivirus naïve adults, children and toddlers in a phase I trial [22].

In a phase II trial, the two formulations were given to flavivirus-naïve individuals. A tetravalent response was observed in 60.0% (F17) and 66.7% (F19) of the participants after a month from the second dose (which was given 6 months following the first dose). Moreover, 97.1% (F17) and 100% (F19) of flavivirus-seropositive individuals showed a response with the same regimen [17]. Similar results were obtained in another phase II trial for flavivirus-experienced individuals. On the contrary, in the naïve group, a response was obtained in 89.0-97.6% individuals (F17) with the lowest response for DENV2 and in 78.0-93.2% (F19) with the lowest response for DENV3. Furthermore, another study revealed that 1 month after the second dose of F17, 56.3% infants and 100% children elicited seroconversion against all serotypes [17].

Inactivated Vaccines – TDENV PIV

Apart from the LAV form of TDEN, GSK and WRAIR has also developed an inactive version, TDENV-PIV. Inactivated vaccines have the advantage of being sufficiently immunogenic while eluding issues like viral interreference and reversal to pathogenic form [22,35]. TDENV-PIV is a purified, tetravalent virus inactivated using formalin [17,24]. 

Phase I trials investigated how two doses (given at 4-week intervals) of TDENV-PIV together with an adjuvant (AS01E or AS03B) performed. In the flavivirus naïve participants, the formulation with AS03B as the adjuvant showed most immunogenicity with all the subjects eliciting a tetravalent response a month after the regime. Nevertheless, this response diminished overtime, remaining detectable in 29% of the participants after a duration of 12 months. On the contrary, the same formulation performed similarly for flavivirus-experienced participants, yet, the antibody levels could be measured even after 3 years from the last dose [17]. This trial portrays that the formulations were tolerated and effective although there is the issue of antibody depletion with time [17,24].

It is important to note that there are a few challenges associated with inactivated vaccines. It can be deduced that due to inactivation with formalin, important viral proteins may have changes. Likewise, due to inactivation it is subjected to reduced replication and hence require multiple doses to achieve long-term immunity. Due to these reasons, it is possible that inactivated vaccine would be useful when used with a booster vaccine. This was supported when the vaccine was successful at providing complete immunity in macaques when used with a live attenuated vaccine booster dose [22]. Lastly, inactivated vaccines are unfortunately more expensive to create in comparison to LAV [36].

Subunit Vaccines

Currently still in the pre-clinical stages, subunit vaccines specialise in avoiding the risk of ADE and also include T-cell protective epitopes [17]. Similar to inactivated virus vaccines, subunit vaccines are safe due to the absence of a nonreplicating virus, hence bypassing viral interference in addition to being safe for the immunocompromised population [36]. However, they require multiple booster doses due to poor immunogenicity and therefore, further research should be done to confirm which combinations of delivery systems and adjuvants would produce the best response [17,22]. 

Structural proteins (like the E-protein) of the DENV which are immunogenic are the foundation of subunit vaccines. E-proteins can be employed as a recombinant form, its individual domains or even through DNA vaccination. Domain III of the E-protein (EDIII) can induce a potent response and hence, is heavily researched recently [19]. In mice and NHPs, recombinant EDIII was seen to elicit serotype specific neutralizing antibodies. When EDIII of all serotypes were combined to make a tetravalent formulation, it was able to induce long-term immunity against all serotypes [19]. 

V180

V180 is the recombinant protein vaccine developed by Merck Sharpe and Dohme (MSD). It comprises a recombinant truncated E-protein belonging to each DENV serotype, formed using the S2 Drosophila cell line [17] [24]. Phase I trials investigated the performance in terms of efficacy and safety of the V180 given in three doses at 1-month intervals. Three different concentrations in combination with two adjuvants, ISCOMATRIX and Alhydrogel, were tested in flavivirus-naïve subjects. Results showed that although all mixtures had a good safety profile, the ISCOMATRIX formulations had better outcomes in terms of immunogenicity. However, this was accompanied by greater adverse effects. Unfortunately, the response reduced overtime with less than half the vaccinated population remaining seropositive after a year from the third dose [17,37]. 

DNA Vaccines

DNA vaccines have the capability to produce non-living, non-replicating antigens that imitate natural infection and hence induce a potent T-cell response in addition to humoral immunity [24]. The composition of DNA vaccines include antigen encoding genes inserted into a plasmid which would eventually be taken up by antigen presenting cells (APCs). Once inside the cells, the antigens are generated and in turn a cytotoxic immune response is induced. However, DNA vaccines have the disadvantage where it only elicits a below satisfactory amount of antibodies and would benefit from the addition of adjuvants to combat this issue [22].

DNA plasmids coding NS1 protein were observed to induce high amounts of anti-NS1 in mice which provided a 100% survival protection rate when challenged with DENV2. Nevertheless, anti-NS1 must be used with caution as it is capable of cross-reacting with endothelial cells and platelets and cause bleeding in mice challenged with it. For this reason, the cross-reactive epitope ∆C was removed to create ∆C NS1 protein. When introduced to anti-∆C NS1, mice did not develop bleeding and endothelial dysfunction [35].

Likewise, monovalent DNA vaccines expressing proteins like prM and E, when introduced to NHPs, showed immunogenicity. Henceforth, truncated E-antigen was chosen to create DNA vaccines as it is similar amongst all DENV serotypes [22]. 

TVDV

The US Naval Medical Research Centre (NMRC) with WRAIR established a tetravalent DNA vaccine. The tetravalent dengue vaccine (TVDV) is a combination of four plasmids that reflect each DENV serotype as it harbours protein (prM and E) encoding genes from each serotype [17].

In a phase I trial, three doses of TVDV along with the adjuvant Vaxfectin were investigated in a flavivirus-naïve population. The results confirmed to be safe although the immunogenicity was insignificant. From the participants that got a high dose formulation, only 29% had measurable levels of antibodies while 79% elicited a DENV specific T-cell response. Nonetheless, it is unknown whether such a response would be beneficial [17,24]. 

Virus like Particle (VLP) Vaccines

VLP based vaccines utilize carriers that are capable of assembling into VLPs that display the antigen on its surface [36]. A recently developed vaccine candidate based on VLP, Dengue Subunit Vaccine Tetravalent (DSV4), portrayed very promising results during pre-clinical testing. The prime focus in DSV4 is EDIII, known to induce potent serotype specific neutralizing antibodies. EDIII of all four DENV serotypes are incorporated together with a HBsAg (hepatitis B surface antigen) VLP to be displayed on its surface. With an adjuvant, this formulation was found to be immunogenic in mice and macaques. Furthermore, it prompted serotype specific neutralizing antibodies against multiple genotypes of each serotype. Lastly, the lack of ADE in-vivo makes is unique to currently available whole virus-based vaccines [26,38].

Viral Vector Vaccines

Formerly, viruses such as adenovirus and alphavirus have been employed as vectors to insert DENV antigen coding genes to help elicit an immune response [39]. Likewise, bivalent formulations have been formed through the insertion of prM and E proteins from two serotypes each (one with DENV1 and 3 and the other with the remaining) into the adenovirus vector, cAdVax. This formulation has resulted in antibodies to each serotype when administered to NHPs. This led to the creation of a tetravalent vaccine (cAdVax-DenTV) which exhibited protection against all serotypes in Rhesus macaques [39] [22]. However, it was noted that prior immunity from exposure (either through infection of adenovirus or measles vaccination) can diminish the replication and hence the immune response. Ultimately, it is proposed that virus vectored vaccines may be suitable as a booster like DNA vaccines [22].

Dengue has resulted in high health burden globally. The ultimate goal is to develop a vaccine that can provide long term protection against all four serotypes and be safe for majority of the population. The live attenuated and inactivated whole virus vaccines are currently the most advanced candidates. They provide broad protection and are safe in comparison to the only approved vaccine, Dengvaxia. Long term studies of safety are required before licencing these candidates to ensure that issues like ADE would not arise. The non-whole virus vaccine candidates have shown low immunogenicity although they pose a good safety profile. Research into ideal adjuvants would shed light towards improving the immunogenicity.

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