4.3. Vaccines under clinical trials

## 4.3.1. CYD-TDV Dengvaxia

VLP allows these vaccines to be usually manufactured large-scale in a cost-effective manner, following current good manufacturing practices. They induce quick and fulminant humoral immune responses by displaying antigens in an ordered and repetitive way. Their particulate nature and dimensions allows an efficient assimilation by dendritic cells (DCs) and transportation to lymph nodes, followed by presentation and induction of optimal immune responses. VLPs are renowned for inducing rapid and strong antibody responses. This trait is attributed to their dense, highly repetitive, quasi-crystalline structures [26], see Dengue vaccine candi-

Antigen Vaccination Developer Preclinical Phase I Phase II Phase III

Sanofi Pasteur X X X X

Takeda X X X

X X

X X

Merck X X

US WRAIR X X

X XX X

dates in Table 1.

CYD Live recombinant based on a yellow fever vaccine 17D backbone

DENV-1-4 prM/E 3 doses

DENV-1,3,4 whole genome, DENV-2 prM/E

154 Dengue Fever - a Resilient Threat in the Face of Innovation

DENV-2 whole genome, DENV-1, -3, -4 prM/E

DENV-1–4 whole genome

Soluble DEN 1/2/ 3/4 prM/E protein

Plasmid DNA expressing DENV 1/2/3/4 prM-E

Purified inactivated DENV or plasmid vector expressing prM/E (prime) and liveattenuated DENV

(boost)

Dengue vaccine candidates; adapted from Kirsten et al. [27].

Table 1. Dengue vaccine candidates.

(0/6/12 months)

2 doses (0/ 90 days)

2 doses (0/ 28 days)

3 doses (0/1/ 2 months)

3 doses (0/1/ 3 months)

1 dose US National

Institutes of Health and Butantan (with licenses to other manufacturers)

GSK/US WRAIR/ Fiocruz

US Naval Medical Research Center

Candidate name/ identifier

TV003/TV005 Tetravalent live, attenuated/ recombinant (whole virus DENV1–3 and recombinant DENV2 in DENV4 backbone)

DENVax Tetravalent live, attenuated/ recombinant (whole virus DENV2 and recombinant DENV1/ 3/4 in DENV2 backbone)

DPIV Tetravalent purified inactivated

TVDVTetravalent "shuffled" prM/E expressed from plasmid vector DNA

DEN-80ETetravalent E protein subunit vaccine

vaccine

vaccine

TLAV-TPIV Heterologous primeboost with liveattenuated tetravalent, live-attenuated vaccine and tetravalent alumadjuvanted purified inactivated vaccine

Sanofi Pasteur's CYD vaccine is a live-attenuated tetravalent chimeric vaccine. In this vaccine, the premembrane and envelope proteins from a wild-type dengue virus corresponding to each of the four serotypes are substituted into the yellow fever (YF) 17D vaccine backbone. A strong neutralizing antibody response to DENV2 was elicited in the first CYD clinical trial in healthy adults, which evaluated only the serotype 2 vaccine strain. Participants previously given YF vaccine seroconverted to all four dengue serotypes [28]. The first licensed dengue vaccine, a live, attenuated, tetravalent dengue vaccine (CYD-TDV; Dengvaxia), has recently been registered in 15 countries as a three-dose immunization schedule administered subcutaneously at 6 month intervals [29]. In the case of Dengvaxia, vaccination of children with no previous infection (seronegative) may mimic an initial infection during the first step in the development of ADE. Because vaccine protection is incomplete and unequal against the four serotypes, a natural infection later in life can complete the sequence of events, causing ADE and severe, lifethreatening dengue fever [30].

Following CYD-TDV introduction, it should be administered as a three-dose series given on a 0-/6-/12-month schedule. However, additional evidence is required in order to determine whether equivalent or better protection may be obtained through simplified schedules. In response to a delay in a vaccine dose for any reason, the vaccine course should be resumed (not restarted), maintaining the 6-month interval between subsequent doses. Given the 12 month duration of the immunization schedule and to enable better vaccine monitoring, countries should have vaccine tracking systems implemented. CYD-TDV is not recommended for use in children under 9 years of age, consistent with current labeling, in view of the association of CYD-TDV with increased risk of hospitalized and severe dengue illness in the 2- to 5-year age group. The target age for routine vaccination should be defined by each country, intended to maximize the vaccination impact and programmatic feasibility of targeting specific age groups. For instance, some countries may present the highest incidence of dengue illness among the adult age population and may consider vaccinating people up to 45 years of age in routine programs. The implementation of a routine CYD-TDV vaccination program at 9 years of age in settings meeting the criteria mentioned above is expected to contribute to a 10–30% reduction in symptomatic and hospitalized dengue illness over 30 years [31], see Table 2. This vaccine will be reviewed further in a separate section since, differently to other vaccines in this section, Dengvaxia has already been registered.

#### 4.3.2. TV003 and TV005 Dengue vaccine

The Laboratory of Infectious Diseases at the U.S National Institutes of Health has evaluated numerous monovalent and tetravalent dengue candidate vaccines to identify candidates with the most acceptable safety, infectivity, and immunogenicity profile. Among these, TV003 is an admixture of four live-attenuated recombinant dengue vaccine candidate viruses (rDEN1D30, rDEN2/4D30, rDEN3D30/31, and rDEN4D30) [36]. Various monovalent candidates were initially tested in Phase 1 trials in order to optimize each of the four vaccine virus strains. Vaccine virus serotypes 1, 3, and 4 are based on complete viruses, while serotype 2 is a recombinant


cells. The DENV-2 PDK-53 virus has proved to be safe, well-tolerated, immunogenic, and elicits long-term humoral and cellular immune responses to DENV-2, based on clinical trials conducted in the United States and Thailand [38]. Three chimeric strains (TDV-1, TDV-3, and TDV-4) were engineered by substituting the premembrane (prM) and envelope (E) structural genes of the respective DENV strains into the attenuated TDV-2 backbone [39]. TDV is designed to promote humoral and cellular protective immune responses against all four dengue serotypes, as it contains the premembrane and envelope proteins unique to each serotype. These specific proteins are needed to induce neutralizing antibodies. The use of DENV-2 as a backbone for TDV may confer additional protection against dengue. In particular, TDV contains the genes encoding the conserved nonstructural (NS) proteins within the dengue backbone; and these proteins have been shown to be important in generating T-cellmediated responses to dengue infection. Furthermore, anti-NS1 antibodies have been associated with cross-protective humoral immune responses [40]. Table 4 shows some of the studies

author /year

Whitehead et al. 2017 [37]

Kirkpatrick et al. 2016 [36]

Conclusion

dengue-endemic areas.

In summary, the authors demonstrated that the NIH tetravalent dengue vaccine TV003 is well-tolerated in flavivirus-experienced individuals and elicits robust post-vaccination neutralizing antibody titers.

Current Status of Vaccines against Dengue Virus http://dx.doi.org/10.5772/intechopen.80820 157

TV003 induced complete protection against challenge with rDEN2Δ30 administered 6 months after vaccination. TV003 will be further evaluated in

The Walter Reed Army Institute of Research (WRAIR) in collaboration with GlaxoSmithKline Vaccines (GSK) developed a live-attenuated tetravalent dengue virus vaccine candidate comprised of four live virus strains representing each of the four DENV types. These strains were attenuated by serial passage in primary dog kidney (PDK) cells [44]. The US Navy Naval Medical Research Center (NMRC) has developed a tetravalent DNA vaccine (TVDV), formulated with Vical's Vaxfectin adjuvant, containing genes encoding the premembrane (prM) and envelope (E) proteins for all four serotypes of dengue virus. Both Vaxfectinformulated and unformulated vaccines are currently being evaluated in Phase I human

Inactivated vaccines are assumed to provide acceptable safety profiles across a wide age range as well as in immunocompromised hosts. In addition, these can be co-administered with other vaccines. Shortened vaccination schedules and rapid immunization are also feasible using this type of vaccines. For these reasons, a safe and efficacious tetravalent DENV PIV could be suitable for national immunization programs across broad age ranges and baseline health

conducted to determine the effectiveness of this vaccine.

4.3.4. DPIV tetravalent purified inactivated vaccine

Reference Lead

Table 3. Some TV003 vaccine safety and immunogenicity studies.

In a randomized trial, the live-attenuated tetravalent dengue vaccine TV003 is well- tolerated and highly immunogenic in subjects with flavivirus exposure

The live-attenuated dengue vaccine TV003 elicits complete protection against dengue in a human

prior to vaccination

challenge model

testing [45].

Table 2. Some CYD-TDV Dengue vaccine safety and immunogenicity studies in different populations.

virus based on the serotype 4 vaccine strain with the structural proteins replaced by those of serotype 2. A single dose of TV005 elicits seroconversion rates above 90% against each serotype, and 90% of flavivirus-naive recipients displayed a tetravalent response. TV003 or TV005 has been licensed to several manufacturers, including Butantan, VaBiotech, and Merck. Phase 2 studies are underway in Brazil and Thailand, and a Phase 3 trial led by Butantan began in February, 2016, in Brazil [27], see Table 3.

#### 4.3.3. DENVax

Takeda's live tetravalent dengue vaccine (TDV) candidate is based on a molecularly characterized attenuated serotype 2 strain (TDV-2). The DENV-2 PDK-53 virus was initially obtained through 53 serial passages of the wild-type (wt) DENV-2 16681 in primary dog kidney (PDK)


Table 3. Some TV003 vaccine safety and immunogenicity studies.

cells. The DENV-2 PDK-53 virus has proved to be safe, well-tolerated, immunogenic, and elicits long-term humoral and cellular immune responses to DENV-2, based on clinical trials conducted in the United States and Thailand [38]. Three chimeric strains (TDV-1, TDV-3, and TDV-4) were engineered by substituting the premembrane (prM) and envelope (E) structural genes of the respective DENV strains into the attenuated TDV-2 backbone [39]. TDV is designed to promote humoral and cellular protective immune responses against all four dengue serotypes, as it contains the premembrane and envelope proteins unique to each serotype. These specific proteins are needed to induce neutralizing antibodies. The use of DENV-2 as a backbone for TDV may confer additional protection against dengue. In particular, TDV contains the genes encoding the conserved nonstructural (NS) proteins within the dengue backbone; and these proteins have been shown to be important in generating T-cellmediated responses to dengue infection. Furthermore, anti-NS1 antibodies have been associated with cross-protective humoral immune responses [40]. Table 4 shows some of the studies conducted to determine the effectiveness of this vaccine.

#### 4.3.4. DPIV tetravalent purified inactivated vaccine

virus based on the serotype 4 vaccine strain with the structural proteins replaced by those of serotype 2. A single dose of TV005 elicits seroconversion rates above 90% against each serotype, and 90% of flavivirus-naive recipients displayed a tetravalent response. TV003 or TV005 has been licensed to several manufacturers, including Butantan, VaBiotech, and Merck. Phase 2 studies are underway in Brazil and Thailand, and a Phase 3 trial led by Butantan began in

Table 2. Some CYD-TDV Dengue vaccine safety and immunogenicity studies in different populations.

Capeding et al. 2011 [35]

author /year

Arredondo-García et al. 2018 [32]

Lee et al. 2018 [33]

Skipetrova et al. [34]

Conclusion

women.

children.

Data from the clinical trials for up to year 4 after first vaccination indicate a positive benefit–risk profile for the CYD-TDV vaccine for the

Given the absence of efficacy and half-life data for any of the second-generation vaccine candidates, it was assumed that NVC is 80% efficacious with a half- life of 8 years.

In the small dataset assessed, no evidence of increased adverse pregnancy outcomes has been identified from inadvertent immunization of women in early pregnancy with CYD-TDV

compared with the control group. The conclusions are limited to vaccination in CYD-TDV in the first trimester, since no data are available on pregnancy outcome for administration of this vaccine in the second or third trimester. The data described here, and those continuing to emerge from the on-going clinical development and post-marketing of CYD-TDV, provide a valuable contribution to the currently limited available information on the use of the dengue vaccine in pregnant

The safety profile of TDV in a flavivirus endemic population was consistent with previous reports from flavivirus-naïve populations. A vaccine regimen of either three TDV vaccinations administered over a year or two TDV vaccinations given more than 8 months apart resulted in a balanced antibody response to all four dengue serotypes in this flavivirus-exposed population, including

population aged 9 years old.

Takeda's live tetravalent dengue vaccine (TDV) candidate is based on a molecularly characterized attenuated serotype 2 strain (TDV-2). The DENV-2 PDK-53 virus was initially obtained through 53 serial passages of the wild-type (wt) DENV-2 16681 in primary dog kidney (PDK)

February, 2016, in Brazil [27], see Table 3.

Reference Lead

Four-year safety follow-up of the tetravalent dengue vaccine efficacy randomized controlled trials in Asia

156 Dengue Fever - a Resilient Threat in the Face of Innovation

Dengue vaccination during pregnancy—an overview

A multi-country study of dengue vaccination strategies with Dengvaxia and a future vaccine candidate in three dengue-endemic countries:

Live-attenuated, tetravalent dengue vaccine in children, adolescents and adults in a dengue-endemic country: Randomized controlled phase I trial in the

Vietnam, Thailand, and Colombia.

and Latin America,

of clinical trials data.

Philippines.

4.3.3. DENVax

The Walter Reed Army Institute of Research (WRAIR) in collaboration with GlaxoSmithKline Vaccines (GSK) developed a live-attenuated tetravalent dengue virus vaccine candidate comprised of four live virus strains representing each of the four DENV types. These strains were attenuated by serial passage in primary dog kidney (PDK) cells [44]. The US Navy Naval Medical Research Center (NMRC) has developed a tetravalent DNA vaccine (TVDV), formulated with Vical's Vaxfectin adjuvant, containing genes encoding the premembrane (prM) and envelope (E) proteins for all four serotypes of dengue virus. Both Vaxfectinformulated and unformulated vaccines are currently being evaluated in Phase I human testing [45].

Inactivated vaccines are assumed to provide acceptable safety profiles across a wide age range as well as in immunocompromised hosts. In addition, these can be co-administered with other vaccines. Shortened vaccination schedules and rapid immunization are also feasible using this type of vaccines. For these reasons, a safe and efficacious tetravalent DENV PIV could be suitable for national immunization programs across broad age ranges and baseline health status, as well as an active immunization option for travelers and military personnel, and a potential tool for outbreak response [46]. Table 5 shows several DPIV vaccine safety and immunogenicity studies.

4.3.5. DEN 80E vaccine

tiveness of this vaccine.

vaccine.

anticipate its potential use as a travel vaccine [52].

This vaccine (developed by Hawaii Biotech and now licensed to Merck) is composed of a recombinant truncated protein corresponding to 80% of the N-terminal DENV E protein (DEN-80E). The C-terminal truncation of the E protein at amino acid 395 removes the membrane anchor sequence of the protein, resulting in a recombinant E protein with improved secretion, purification and immunogenicity. The DEN-80E protein for each of the four dengue serotypes has been expressed in the Drosophila S2 expression system to produce a tetravalent vaccine [49], which induces a high level expression of proteins of interest. Specifically, the system was chosen to express a plasmid containing the prM and N-terminal 80% of the E gene sequence of DENV-2. The resulting polyprotein undergoes cleavage by endogenous proteases and the 80E protein with a native-like N terminus is released. Two doses of the DENV-2 subunit 80E protein were administered to rhesus macaques in combination with one of seven different adjuvants at a 3-month dosing interval. Following this administration, animals were challenged with wild-type DENV-2 2 months after the last dose of vaccine. Neutralizing antibodies were detected in all study animals after the first dose and this response was boosted by the second dose. The highest neutralizing antibody titers were produced by the r80E protein formulated with the adjuvants AS05 or AS08, and protection against viremia was correlated with a higher neutralizing antibody titer at challenge. The same system was employed to generate recombinant subunit E proteins (80E) of the other DENV serotypes. A tetravalent formulation of the recombinant 80E proteins was evaluated in mice and nonhuman primate experiments. In some instances, the NS1 protein of DENV-2 was included in the formulation to potentially enhance the immune response to the vaccine. Macaques were immunized with the tetravalent formulation four times (day 0, 28, 67, and 102) and were challenged 5 months after the last dose. Due to the limited number of monkeys in each group, monkeys were only challenged with DENV-2 or DENV-4. Monkeys developed a robust neutralizing antibody response to all four DENV serotypes and were completely protected from DENV-2 challenge [50]. Table 6 shows some of the studies conducted to determine the effec-

Current Status of Vaccines against Dengue Virus http://dx.doi.org/10.5772/intechopen.80820 159

4.3.6. TVDV tetravalent "shuffled" prM/E expressed from a plasmid vector DNA vaccine

The U.S. Naval Medical Research Center (NMRC) developed a tetravalent plasmid DNA vaccine candidate using prM and E protein genes expressed in a plasmid vector. A DENV-1 monovalent candidate of this vaccine was evaluated for safety and immunogenicity through a phase I clinical trial on healthy flavivirus-naïve adults using a three-dose schedule at 0/1/ 5 months. The results showed poor immunogenicity. Although it is possible that TVDV may have a role as a travel vaccine in the future, the available data is currently insufficient to

The TVDV is a mixture of equal amounts of four monovalent double-stranded plasmid DNA vaccines produced under current Good Manufacturing Practices conditions in the United States. Each monovalent plasmid contains the prM and E genes of dengue 1, 2, 3, or 4 viruses cloned into the backbone plasmid VR1012 (Vical Incorporated, San Diego, CA) [53]. Table 7 shows some of the studies conducted to determine the effectiveness of this


Table 4. Some TDV(DENVax) vaccine safety and immunogenicity studies.


Table 5. Some DPIV vaccine safety and immunogenicity studies.

#### 4.3.5. DEN 80E vaccine

status, as well as an active immunization option for travelers and military personnel, and a potential tool for outbreak response [46]. Table 5 shows several DPIV vaccine safety and

author /year

George et al. 2015 [41]

Osorio et al. 2014 [42]

Osorio et al. 2011 [43]

author /year

Diaz et al. 2018 [47]

Lepine et al. 2017 [48]

Conclusion

development.

worldwide.

Conclusion

of DPIV clinical development.

this dengue vaccine candidate.

All DPIV formulations were well-tolerated. No vaccine-related serious adverse events were observed through 12 months after the second vaccine dose. In all DPIV groups, geometric mean antibody titers peaked at Day 56, waned through 6 months after the second vaccine dose, and then stabilized. In the nine subjects where boosting was evaluated, a strong anamnestic response was observed. These results support continuation of the clinical development of

TDV was generally well-tolerated, induced trivalent or broader neutralizing antibodies to DENV in most flavivirus-naive vaccines, and is undergoing further

The authors emphasize the acceptable tolerability and immunogenicity of the tetravalent DENVax formulations in healthy, flavivirus-naive adults. Further clinical testing of DENVax in different age groups and in dengue-endemic areas is warranted.

The DENVax vaccine is considerably different from previously tested tetravalent vaccines in that all four strains contain the same attenuating mutations as the DEN-2 PDK-53 strain, a strain that has been shown to be both safe and immunogenic in humans. Such vaccine is critically needed to protect people from the threat of dengue infection and improve public health

Results from this first phase I study of a new vaccine candidate with inactivated DENV in a dengueprimed population showed that all four DPIV vaccine formulations were well-tolerated and immunogenic. This new investigational DPIV vaccine had an acceptable safety profile in a small number of flavivirus-primed healthy adult subjects and all formulations boosted neutralizing antibodies (Nab) responses, with complex adjuvants increasing immunogenicity versus alum adjuvantation. Nab titers remained high (and above baseline titers) through M13. These results encourage continuation

immunogenicity studies.

clinical trial

against dengue fever

Puerto Rico.

United States

Reference Lead

158 Dengue Fever - a Resilient Threat in the Face of Innovation

Safety and immunogenicity of a live-attenuated tetravalent dengue vaccine candidate in flavivirusnaive adults: a randomized, double-blinded Phase 1

Safety and immunogenicity of a recombinant liveattenuated tetravalent dengue vaccine (DENVax) in flavivirus-naive healthy adults in Colombia: a randomized, placebo-controlled, phase 1 study

Development of DENVax: A chimeric dengue-2 PDK-53-based tetravalent vaccine for protection

Table 4. Some TDV(DENVax) vaccine safety and immunogenicity studies.

Reference Lead

Phase I randomized study of a tetravalent dengue purified inactivated vaccine in healthy adults from

Phase 1 randomized study of a tetravalent dengue purified inactivated vaccine in healthy adults in the

Table 5. Some DPIV vaccine safety and immunogenicity studies.

This vaccine (developed by Hawaii Biotech and now licensed to Merck) is composed of a recombinant truncated protein corresponding to 80% of the N-terminal DENV E protein (DEN-80E). The C-terminal truncation of the E protein at amino acid 395 removes the membrane anchor sequence of the protein, resulting in a recombinant E protein with improved secretion, purification and immunogenicity. The DEN-80E protein for each of the four dengue serotypes has been expressed in the Drosophila S2 expression system to produce a tetravalent vaccine [49], which induces a high level expression of proteins of interest. Specifically, the system was chosen to express a plasmid containing the prM and N-terminal 80% of the E gene sequence of DENV-2. The resulting polyprotein undergoes cleavage by endogenous proteases and the 80E protein with a native-like N terminus is released. Two doses of the DENV-2 subunit 80E protein were administered to rhesus macaques in combination with one of seven different adjuvants at a 3-month dosing interval. Following this administration, animals were challenged with wild-type DENV-2 2 months after the last dose of vaccine. Neutralizing antibodies were detected in all study animals after the first dose and this response was boosted by the second dose. The highest neutralizing antibody titers were produced by the r80E protein formulated with the adjuvants AS05 or AS08, and protection against viremia was correlated with a higher neutralizing antibody titer at challenge. The same system was employed to generate recombinant subunit E proteins (80E) of the other DENV serotypes. A tetravalent formulation of the recombinant 80E proteins was evaluated in mice and nonhuman primate experiments. In some instances, the NS1 protein of DENV-2 was included in the formulation to potentially enhance the immune response to the vaccine. Macaques were immunized with the tetravalent formulation four times (day 0, 28, 67, and 102) and were challenged 5 months after the last dose. Due to the limited number of monkeys in each group, monkeys were only challenged with DENV-2 or DENV-4. Monkeys developed a robust neutralizing antibody response to all four DENV serotypes and were completely protected from DENV-2 challenge [50]. Table 6 shows some of the studies conducted to determine the effectiveness of this vaccine.

#### 4.3.6. TVDV tetravalent "shuffled" prM/E expressed from a plasmid vector DNA vaccine

The U.S. Naval Medical Research Center (NMRC) developed a tetravalent plasmid DNA vaccine candidate using prM and E protein genes expressed in a plasmid vector. A DENV-1 monovalent candidate of this vaccine was evaluated for safety and immunogenicity through a phase I clinical trial on healthy flavivirus-naïve adults using a three-dose schedule at 0/1/ 5 months. The results showed poor immunogenicity. Although it is possible that TVDV may have a role as a travel vaccine in the future, the available data is currently insufficient to anticipate its potential use as a travel vaccine [52].

The TVDV is a mixture of equal amounts of four monovalent double-stranded plasmid DNA vaccines produced under current Good Manufacturing Practices conditions in the United States. Each monovalent plasmid contains the prM and E genes of dengue 1, 2, 3, or 4 viruses cloned into the backbone plasmid VR1012 (Vical Incorporated, San Diego, CA) [53]. Table 7 shows some of the studies conducted to determine the effectiveness of this vaccine.


DENV serotypes were able to induce neutralizing antibodies and protect against viral challenge. DENV-1 and DENV-2 monovalent candidates have also been evaluated in NHPs. Monkeys were immunized subcutaneously with four doses of the monovalent vaccine (50– 100 g protein per dose, formulated in Freund's adjuvant), which proved to be immunogenic and provided protection against viral challenge. Adjuvants suitable for human use are under evaluation, including N. meningitidis serogroup A capsular polysaccharide (CPSA) adsorbed

year

Thomas et al. 2018 [53]

Raviprakash et al. 2012 [54]

Conclusion

TVDV-Vaxfectin was safe and well-tolerated in this early Phase 1 human clinical trial. Whereas antidengue IFNγ T-cell responses occurred in most of the study subjects, anti-dengue neutralizing antibody responses were poor. Utilization of alternative delivery methods as well as examining prime-boost approaches may result in a more robust and long-lasting humoral immune response.

Current Status of Vaccines against Dengue Virus http://dx.doi.org/10.5772/intechopen.80820 161

The formulated vaccine and the adjuvant were tested for safety and/or immunogenicity in New Zealand white rabbits using a repeat dose toxicology study. The formulated vaccine and the adjuvant were found to be well-tolerated by the animals. Animals injected with formulated vaccine produced strong neutralizing antibody response to

all four dengue serotypes.

Finally, we want to reflect on the implications of the co-circulation of the dengue virus and the

First, we will analyze the fact that the appearance of the infection by the Zika virus (another flavivirus) in zones of high prevalence for dengue constitutes an interesting challenge for the

Among pathogenic human flaviviruses, DENV and ZIKV are most closely related to each other, with 55.1–56.3% amino acid sequence identity. Zika virus is closer to dengue virus than to any of the other flaviviruses and indeed is almost close enough to think of it as a fifth serotype [10]. Accordingly, emerging literature indicates many similarities between these two viruses in terms of interactions between the virus and host immune system. For both viruses, the interferon system is the central mediator of host defense and target of a viral counterattack,

Zika virus, as well as on the new indications for the use of the Dengvaxia vaccine.

5.1. Zika virus infection means new challenges in dengue vaccine development

on aluminum hydroxide [25].

development of the ideal vaccine for both viruses.

Reference Lead author/

Table 7. Some TVDV vaccine safety and immunogenicity studies.

Safety and immunogenicity of a tetravalent dengue DNA vaccine administered with a cationic lipidbased adjuvant in a Phase 1 clinical trial

A dengue DNA vaccine formulated with Vaxfectin® is well-tolerated, and elicits strong neutralizing antibody responses to all four dengue serotypes in New Zealand white rabbits

5. Final thoughts

Table 6. Some DEN 80E Vaccine safety and immunogenicity studies.

#### 4.4. Vaccine candidates under preclinical assays

There are numerous vaccine candidates that are being studied in preclinical trials, as can be seen in Table 8.

#### 4.4.1. EDIII-p64k fusion proteins and EDIII-capsid fusion proteins expressed in E. coli

Te Pedro Kourí Tropical Medicine Institute (IPK) in collaboration with the Center for Genetic Engineering and Biotechnology (CIGB) in Cuba have led the development of various recombinant subunit vaccine candidates. One approach is based on fusion of DENV EDIII to the carrier protein p64k of Neisseria meningitidis, and this EDIII-p64k fusion protein is then expressed in E. coli. Evaluations in mice showed that monovalent vaccine candidates for all


Table 7. Some TVDV vaccine safety and immunogenicity studies.

DENV serotypes were able to induce neutralizing antibodies and protect against viral challenge. DENV-1 and DENV-2 monovalent candidates have also been evaluated in NHPs. Monkeys were immunized subcutaneously with four doses of the monovalent vaccine (50– 100 g protein per dose, formulated in Freund's adjuvant), which proved to be immunogenic and provided protection against viral challenge. Adjuvants suitable for human use are under evaluation, including N. meningitidis serogroup A capsular polysaccharide (CPSA) adsorbed on aluminum hydroxide [25].
