Dengue Vaccines - Ongoing Challenges and Current

Prévia do material em texto

Dengue Vaccines: Ongoing Challenges and Current 
Status in the Advancement of Different Candidates
Muhammad Hassan, Ali Hassan, Muhammad Farooq, Samia Afzal,* Mohsin Ahmad Khan, 
Iram Amin, Muhammad Shahid, Muhammad Idrees, & Ahmad Ali Shahid
Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan
*Address all correspondence to: Samia Afzal, Division of Molecular Centre of Excellence in Molecular Biology (CEMB), 87 West Canal Bank 
Road Thokar Niaz Baig, University of the Punjab, Lahore, Pakistan; Tel.: +92-42-5293141; Fax: +92-42-5293149, E-mail: samiaraza@live.com
ABSTRACT: Dengue is a vector-borne highly systemic infectious disease of the tropical and subtropical countries 
and is devastating millions of lives worldwide. It may be self-eliminated like a mild fever or may cause life-threaten-
ing fatal complications as dengue hemorrhagic fever and dengue shock syndrome. The lack of specific and effective 
antiviral drugs and vaccines amplify its transmission rate across the world. The development of the dengue vaccine 
has been an ambitious task due to the presence of four different dengue serotypes capable of carrying antibody en-
hancement complex mechanisms. In this review, we have summarized the ongoing challenges in the construction of a 
dengue vaccine and the current status of the vaccine development. Limited knowledge of immune responses against 
dengue infection, lack of human or animal model of disease, and suboptimal assay strategies to detect immune re-
sponses after infection or vaccination, are some barriers to vaccine and drug development. A tetravalent vaccine with 
low cost, high efficiency, and capable of eliciting immune responses against all four serotypes is needed to minimize 
the epidemics. Currently, only one live attenuated chimeric dengue vaccine, the CYD Dengue Vaccine, has completed 
its third phase and has been licensed. DENVax and TetraVax-DV-TV003 (TV003) are in the third phase while others 
are still in the first trial phase. 
KEY WORDS: aedes mosquito, chimeric vaccine, dengue virus, endemic, public health
I. INTRODUCTION
Dengue is the most prevalent systemic arboviral 
infection brought about by one of the four sero-
types (DENV-1 to DENV-4), affecting almost half 
of the world’s population annually.1,2 Dengue is 
transmitted by a mosquito vector, Aedes aegypti, 
which continues to spread throughout the world 
and adopts new habitats due to climatic fluctua-
tions and increased urbanization. The secondary 
vector A. albopictus is also increasing its zone of 
habitat.3 Dengue has crossed malaria concerning its 
nature of the most fast-spreading mosquito-borne 
infectious disease. All four serotypes causing ill-
ness and dengue infection can cross immunolog-
ically result in cross immunization against each 
other for up to two years.4 A patient carrying a 
primary infection is more vulnerable to second-
ary infection with severe dengue hemorrhagic 
fever (DHF) and dengue shock syndrome (DSS) 
due to a phenomenon called antibody-dependent 
enhancement.5 Similarly, infants with high titer 
of maternal dengue antibodies are more prone to 
secondary infection including severe DHF and 
DSS.6 Different serotypes result in different ma-
jor complications like primary infection caused 
by DENV-1 and DENV-3 is associated with more 
severe disease than primary infection of DENV-2 
and DENV-4. Similarly, secondary infection of 
DENV-2 results in severe disease while less se-
vere in case of DENV-4 as depicted in Table 1.7,8
Dengue mostly occurs in tropical and subtrop-
ical areas. Factors such as heavy rainfall, floods, 
fluctuation in temperature, urbanization, and dis-
tribution of DENV carrier Aedes aegypti strongly 
influence its local spatial variation in transmission.9 
Infection with any serotypes of DENV either results 
in asymptomatic disease or causes a broad spectrum 
of infections ranging from mild fever to fatal DHF 
and DSS. Due to the low efficacy of insecticidal 
strategies10 and lack of antiviral treatment, a dengue 
vaccine development has been following up to break 
the chain of transmission and shrink the economic 
burden of dengue worldwide.11
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8 Hassan et al.
II. GENOMIC STRUCTURE
The capsid of a DENV encloses a single-stranded, 
positive-sense RNA genome of about 10,600 nu-
cleotides. The DENV genome possesses a cap of 
m7G5′ppp5′A but lacks a polyadenylated 3′-tail se-
quence.12 The untranslated regions (UTRs) of both 
5′- and -3′ ends regulate the translation and genomic 
RNA synthesis mechanism.13 As the DENV genome is 
a positive sense RNA carrying an open reading frame, 
it is directly translated into a long single polypeptide 
that is cotranslationally cleaved by cellular and viral 
proteases producing three structural and at least seven 
non-structural proteins as shown in Fig. 1.14 The struc-
tural proteins like capsid, precursor membrane protein 
(prM), and envelope (E) proteins are encoded by the 
amino terminus of the genome while the nonstructural 
proteins are expressed by the remaining sequences.15 
The capsid protein is constructed from 120 amino acids 
and it helps in the packaging of new progeny viruses 
by forming a nucleocapsid. The prM carrying ~ 165 
amino acids and E with ~ 495 amino acids function 
as glycoproteins each possessing two transmembrane 
helices. The prM protein may regulate the folding and 
assembly of the E protein by functioning as a chaperon 
before its cleavage during particle maturation. The E 
protein possess a cellular receptor-binding site(s) and 
a fusion peptide that helps in the attachment and entry 
inside the host cell.16–18
III. OUTBREAKS
The dengue endemic is affecting the tropical and 
subtropical areas of more than 100 countries. Den-
gue fever has been a great challenge for centuries. 
The first observed symptoms showing high compati-
bility with dengue fever were marked down in a Chi-
nese medical encyclopedia (922 AD).19 The disease 
was then named “poison water” and was linked with 
flying insects.20 The dengue epidemics in the USA 
was started in the early 20th century followed by 
the major endemics that occurred in Philadelphia in 
1780.21,22 It is very difficult to ascertain the true im-
pact of dengue globally, but some researchers have 
reported 50 million to 200 million dengue infections, 
500,000 episodes of severe dengue (DHF/DSS), and 
more than 20,000 dengue deaths occur annually.23,24
In Asia, the first report of DHF was received in 
the 1950s in Thailand and the Philippines. However, 
the disease spread throughout Southeast Asia in the 
next 20 years, but by the mid-1970s, the disease was 
hospitalized and caused the death of many new-
borns. The regular epidemics of every 3–5 years of 
duration intensified the dengue transmission in hy-
perendemic areas in the 1980s and 1990s. 
TABLE 1: An association of dengue disease severity 
with all four serotypes
Serotype Major complications
DENV-1 Its primary infection is associated 
with more severe disease than that 
with DENV-2 and DENV-4.
DENV-2 Secondary infection is associated with 
severe disease.
DENV-3 Severe disease with primary infection 
than that of caused by DENV-2 or 
DENV-4. Secondary infection is 
twice that in DENV-4.
DENV-4 Associated with less severe disease.
FIG. 1: Genomic structure of the dengue virus, a positive-sense RNA about 11 kb in size expressing three structural 
and seven non-structural proteins
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Challenges and Current Status of Dengue Vaccines 9
In Pakistan, the first outbreak of DENV-2 com-
menced in 1994 reported by Aga Khan University 
Hospital, Karachi, Pakistan.25 Variations in climate 
and population growth of the urban centers dis-
tracted the developing countries. Several outbreaks 
occurred in South Asia, especially in Sri Lanka, In-
dia, Bangladesh, and Pakistan, but the outbreak of 
DHF that occurred in Punjab, the highly populated 
Province in Pakistan, during the end of the last de-
cade, was a major threat to human health.26 Another 
sudden outbreak of DHF in Karachi increased the 
number of victims during 2005.27 In Faisalabad, 
DHF affected over 4000 individuals and killed hun-
dreds of infected patients during 2011.28 A major de-
structive dengue outbreak was reported in Lahore, 
Punjab, Pakistan in 2011. More than 23,000 people 
were admitted to hospitals, and almost 360 people 
passed away, during which DEN-2 and DEN-3 were 
recognized as predominant serotypes.29 In 2013, a 
severe incidence occurred in Swat, Pakistan, hospi-
talized thousands and killed about 57 patients.30 In 
2017, the same outbreak in Peshawar, Pakistan, also 
hospitalized thousands of patients and killed about 
70 people.31
IV. VACCINE DEVELOPMENT CHALLENGES
The development of a dengue vaccine has been a 
hard-fought and ambitious procedure for decades. 
The unavailability of an appropriate animal model 
and insufficient basic knowledge of correlates, both 
for disease pathogenesis and the host immune re-
sponses have hampered the development of a dec-
orous immunogenic dengue vaccine.32 But, the 
interaction among four serotypes is considered as 
the most catchy and biggest obstacle in the dengue 
vaccine development. As we require an ideal dengue 
vaccine, a tetravalent live-attenuated vaccine (LAV) 
constructed from a mixture of all four serotypes, each 
constituent would need independently to boost up a 
specific monotypic immune response against their 
respective serotype. This strategy has, unfortunately, 
been tested to be hard to complete successfully.33
The mutual and effective interaction of innate 
and adaptive immune responses that bring about 
protection or regulate the pathogenesis of DENV 
infection is partially comprehended.32 A major 
concern with dengue vaccine development is the 
insight of antibody-dependent enhancement mech-
anism, established following primary infection with 
a heterotypic DENV.32,34 The lack of a dengue ani-
mal disease model is the significant factor that ad-
versely affects the immune correlates of protection. 
Nevertheless, immunological and clinical responses 
to DENV infection were studied by using human-
ized small animal models, yet, the results are not as 
concise as to offer a detailed view of in vivo human 
dengue infection pathology and the consequent im-
mune responses.35–38 In addition to the absence of an 
animal model, the dengue human infection model 
is also not present that would support drug and 
vaccine development procedures, immunological 
studies, and the observation of immune correlates. 
Another issue affecting the dengue vaccinology is 
the absence of assays used in the measurement of 
vaccine immunogenicity during preclinical and clin-
ical trials.39
V. IDEAL VACCINE
The primary characteristics of an ideal DENV vac-
cine comprise zero significant reactogenicity, elicita-
tion of antibodies neutralization, and cell-mediated 
long-lasting immunity against all four serotypes. 
As DENV endemic mostly affects the developing 
countries that lack health facilities, that’s why the 
vaccine must be economical with no-repeat booster 
doses.40 An ideal vaccine would be genetically sta-
ble, economical, environmentally friendly, and eas-
ily accessible to all people. Simple storage methods, 
easy transportation, and proper maintenance are also 
important features for an ideal vaccine.41 
A. Vaccine Candidates in Development
Dengue vaccine candidates are classified according 
to their pattern of construction as shown in Fig. 2.
B. Replicating Viral Vaccine or LAV
1. Chimeric Dengue Vaccine
It is a recombinant form of the YF17D vaccine 
in which the prM/E RNAs of the YF17D was 
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10 Hassan et al.
substituted with the corresponding receptor se-
quences of all dengue serotypes.42 CYD-TDV is 
the only one of the three LAVs, who has completed 
its Phase III trials with a long-term follow-up of 5 
years, others are still in late-stage development (Ta-
ble 2). It was designed by a French multinational 
pharmaceutical company, Sanofi Pasteur, and gave 
it Dengvaxia as a trade name.33 Clinical trials of 
CYD-TDV has shown its high efficacy among pa-
tients over 9 years old. The immunity boosted up 
by CYD-TDV lasts up to 4 years, and some factors 
like serotype, age, dengue sera status of the patient 
before vaccination appear to affect the vaccine effi-
cacy. Its Phase III trial has shown its high efficacy 
and was the first dengue vaccine to be licensed in 
2015.43 More than 180 countries have approved 
CYD-TDV and its first mass immunization cam-
paign was started in the Philippines and Brazil in 
2016 and 2017.44 In 2019, the U.S. Food and Drug 
Administration (FDA) has approved Dengvaxia to 
be used against seropositive individuals 9–16 years 
of age living in the dengue-affected areas of the 
United States. The European Medicines Agency 
also recommended this vaccine for seropositive in-
dividuals only.33
2. Current Limitations in the Adoption of the 
CYD-TDV Vaccine
It has been noted that critical issues arise due to sero-
type intervention that should be inscribed by Sanofi 
Pasteur. By administrating the vaccine as a tetrava-
lent formulation the imbalances in viral replication 
were observed that included four serotypes (mon-
ovalent) along with immune dominance linked with 
the epitopes.45 It was observed that CYD has shown 
no protection against DENV-2.46 Furthermore, it 
was observed that only modest protection could be 
provided by the vaccine to the uninfected individu-
als in Latin America (60.8%) and Asia (56.5%). The 
efficacy of the vaccine was reported to be the lowest 
against DENV-2 (42.3% and 35%) respective to the 
regions described.47,48 The reduced efficacy of the 
vaccine for dengue naïve individuals is still under 
question. Long-term follow-up showed that the risk 
of hospitalization was lower as compared to pla-
cebo and higher in the recipients of CYD in their 
post-vaccination period.40
In addition, it could only be administered to the 
individuals of a specific range of age, 9–45 years.49 
Countries including Mexico, Singapore, Brazil, and 
the Philippines have been known to grant market ac-
cess to vaccines. However, due to some unreliability 
regarding partial protection provided by CYD, it is 
facing limitations globally.50
Takeda has developed the DENVax vaccine 
that contains DENV-2, whereas the chimeric vi-
ruses contain prM as well as the E proteins of 
DENV-1, DENV-3, and DENV-4 are inserted into 
the DENV-2 backbone. Due to the DENV-2 back-
bone, the NS proteins are also present, unlike the 
Dengvaxia. These NS proteins may be essential to 
generate antibodies against NS1 and T-cell medi-
ated responses.51 The vaccine has worked well in the 
previoustrials in humans and non-human primates, 
by synthesizing high amounts of neutralizing anti-
bodies against all serotypes as well as by generating 
T-cell mediated responses. These may be essential 
for the protection from dengue fever.52 The AG129 
mice model was selected to investigate the effect of 
maternal DENVax vaccination on the re-inoculation 
effects in offsprings. It was observed that the pups 
born by immunized mothers also carry maternal 
antibodies.53
FIG. 2: Classification of dengue vaccines
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Challenges and Current Status of Dengue Vaccines 11
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3. Cell Culture Passage–Based LAV
Initially, Mahidol University Thailand, started the 
development of LAV by the process of serial passag-
ing in cell lines. Attenuation of all DENV serotypes 
was done to form a tetravalent formulation but the 
vaccine could not evoke a well-balanced immune 
response due to which the vaccine was considered to 
be a fail.54,55 Also, the reactions observed were even 
worse, including myalgia, fever, retro-orbital pain, 
and rash. The adverse reactions were observed to be 
a result of the DENV-3 strain. Due to these reasons, 
the further development of LAV was stopped.56 Wal-
ter Reed Army Institute of Research in the United 
States, in collaboration with GlaxoSmithKline, also 
developed another LAV which is under the process 
of clinical trials. Serotypes passaging to attenuate 
DENV was done in the primary dog kidney cells.57 
However, the terminal passaging was carried out 
in the fetal rhesus lung cells. To observe the can-
didate’s level of attenuation and immunogenicity 
balance, the evaluation of monovalent vaccine can-
didates was done in rhesus macaques. In the case 
of humans, the DENV-1 was found to be most re-
actogenic as compared to the other three which 
showed mild reactivity. Among all the serotypes, the 
vaccine against DENV-1 was under-attenuated and 
DENV-4 was over-attenuated.58,59 However, to im-
prove the immunogenicity, the vaccine components 
were changed and replaced by a lower passaged vi-
rus (PDK-6 rather than PDK-20) and by a higher 
passaged virus (PDK-27 rather than PDK-20) in 
DENV-4 and DENV-2, respectively.
4. Site-Directed Mutagenesis–Based LAV
This methodology was first effectively investigated 
by the Laboratory of Infectious Disease at the Na-
tional Institute of Allergy and Infectious Disease 
(NIAID), National Institutes of Health (NIH), 
Bethesda, MD, USA. NIH has set up generic per-
mits with producers in Brazil (Instituto Butantan), 
Vietnam (Vabiotech), and India (Serum Institute 
of India and Panacea Biotech) for its production. 
This vaccine is a combination of four DENV strains 
weakened by site-guided mutagenesis to erase 30 
nucleotides in the 3ʹ UTR. DENV-1 and DENV-4 
weakened strains were assigned as DEN1Δ30 and 
DEN4Δ30, individually.60 Since the DEN2∆30 and 
DEN3∆30 infections were not appropriate candi-
dates, so these weak strains were made by utilizing 
DEN4Δ30 and supplanting their primary prM and 
E qualities with those of the comparing serotype. 
There is a low oral infection for A. aegypti mosqui-
toes of both DEN3/4∆30 and DEN2/4∆30 vaccine 
candidates.61,62
It is seen that DEN2/4∆30 vaccine is immuno-
genic and safe when the dosage of 103 pfu given in 
Phase I trial,63 and clinical trials of DEN3/4∆30 are 
ongoing. Strains of rDEN3/4Δ30 and rDEN2/4Δ30 
show over attenuation which is a result of chime-
rization. The modification was done in the DENV-3 
part and 31 nucleotides were removed from rDEN3 
Δ30 of strain rDEN3Δ30/31.64 Now, Brazil is the 
country where Phase III of this vaccine candidate 
has started.65 About 30 mutations in The DENV-4 
and DENV-1 did not show any symptoms and is also 
safe and immunogenic at 103 pfu. Few symptoms 
like rashes, rise in with a faint rash, alanine amino-
transferase (ALT) enzyme levels in some persons, 
and leukopenia in 7–40% were observed at a more 
dose of the vaccine of 105 pfu for DEN4∆30.62,66,67
C. Non-Replicating Viral Vaccine or 
Inactivated Vaccine
Based on their inactivated or non-replicating nature, 
immunity develops without the chance of getting the 
infection. Vaccines belonging to this class can be de-
veloped by different approaches like VLPs, subunit 
proteins, DNA vaccines, and many more.
(i) Advantages of this vaccine include: better 
immune response in case of tetravalent formulation, 
low chances of negative reactions,and appropriate 
for immune-compromised persons.
(ii) Disadvantages include: adjuvants are re-
quired and this vaccine may result in ADE. Spectrum 
is also not a broad less potent immune response.68,69
1. Purified Inactivated Virus (PIV)
These vaccines provide protection against live 
pathogens and are composed of inactivated material 
from a pathogen (like bacteria or virus).68 Immunity 
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Challenges and Current Status of Dengue Vaccines 13
was stimulated by using inactivated Dengue vaccine 
which uses protein components like C, M, E, and 
NS1 as antigens, but rather using single-type vac-
cines, composite vaccines stimulate better protec-
tion. Compared with inactivated vaccines are much 
protective than LAVs, as they have better controlled 
immune balance and there is no hidden vulnerability 
of reactivation.79
Inactivated viral vaccine is much safer than oth-
ers and it’s the most striking advantage of this vac-
cine. Additionally, viral interference can be avoided 
between live viruses in the tetravalent formulation. 
But, the formation of inactivated dengue virus is 
hampered by low immunogenicity. Presently, not 
a large number of purified formalin-inactivated 
(PIV) are in clinical trials. In Phase I, a vaccine is 
evaluated which is developed by WRAIR named 
DENV-1 PIV, having aluminum hydroxide as adju-
vant. When the vaccine was given to some volun-
teers, the results demonstrated that the vaccine was 
immunogenic and safe. These results were obtained 
by measuring the antibody responses by neutraliza-
tion tests and ELISA.80
The efficiency of inactivated viral vaccine can 
be enhanced by adding adjuvants WRAIR devel-
oped a purified, inactivated DENV-2 vaccine and a 
DENV-1 equivalent will enter clinical trials in the 
near future.68 Certified Vero cells were used for the 
propagation of viruses to prepare the inactivated 
vaccine candidates. Further, these propagated vi-
ruses were then subjected to ultrafiltration for con-
centration and finally, sucrose gradients were used 
for purification.69 Immunogenicity is not present in 
all DEN viruses, a strain of DEN-I PIV after two 
doses depict the same immunogenicity in mon-
keys. Immunogenicity, protection, and purity of 
DEN-2 PIV WR-4 showing competitive results to 
other PIVs made for Japanese encephalitis flavivi-
ruses and tickborne encephalitis utilizing related 
procedures.68
2. Recombinant Subunit Dengue Vaccine
In this approach, the E protein present in the enve-
lope of dengue virus has been selected for the devel-
opment of recombinant protein vaccine. E protein 
expression is linked with expression of prM protein 
that may work as chaperone for the correct folding 
of E protein. Additionally, prM-E protein that ex-
pressed intracellularly will be cleaved by cellular 
furin into prM and E protein. Studies have demon-
strated that, if at N terminal 80% E protein (r80E) 
was expressed, then it assisted the E protein secre-
tion in cultured cell.81 In fruit fly cells, r80E pro-
tein of dengue virus-2 was expressed successfully 
and its high yield was purified and formulated with 
AS04, AS05, and AS06 adjuvants as the subunit 
DENV vaccine. Rhesus macaques was immunized 
with the r80E subunit vaccine which neutralizes the 
antibodies and protects against viremia.82 E protein 
of dengue virus consists of three domains I, II and 
III which are structurally distinct.83 For vaccine de-
velopment, the most important domain of E protein 
is domain III (EDIII). This domain plays a vital role 
for viral entry into host cell by binding to the host 
cell receptor84,85 and to develop long time immunity 
against dengue virus domain III also helps to pro-
duce serotype-specific antibodies. Domain III along 
with the ability of binding sites also contains the 
ability of independent folding as individual struc-
ture.86 From the consensus sequence of domain III to 
all four types of dengue virus, domain III was also 
produces and this obtained domain III has ability to 
neutralize the antibodies against all four serotypes 
of dengue virus in mice.87 Bio-hazard is a problem 
associated with LAV and inactivated vaccine and 
this problem can be overcome by using protein vac-
cine as no bio-hazard is associated with it. Addition-
ally, use of protein subunit vaccine is easy, safe and 
inexpensive.88 
3. Dengue DNA Vaccine
Vaccines that consist of plasmids containing den-
gue virus genes are known as DNA vaccines and 
are reproduced in bacteria, e.g., Escherichia coli. 
A eukaryotic promoter and termination sequence is 
present in these plasmids to transcribe viral genes. 
DNA-based vaccines have potential advantages of 
easy production, stability, ability to add new genes, 
and ability to immunize against multiple pathogens 
with a single construct. DNA vaccine contains a 
plasmid consist of an antigen gene. An organism 
gets immunized with this vaccine when antigen 
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present on plasmid was taken up by antigen-pre-
senting cells (APCs). Once the plasmid enters the 
cell, the antigen was encoded and displayed on the 
surface of MHC class I molecules and initiates a cy-
totoxic immune response. Naval Medical Research 
Center (NMRC), USA, has developed a DENV-1 
DNA vaccine candidate (D1ME100) in which prM 
and E gene of DENV-1 serotype was cloned into a 
plasmid vector, which was then immunized in mice 
and macaques without adjuvant89 before Phase I tri-
als in healthy adults. Although the DENV-1 DNA 
vaccine was found to be effective, the neutralizing 
antibody titers and the number of responders were 
found to be low.89,90 
DNA vaccines are more beneficial than conven-
tional vaccines due to their stability, transportation 
at room temperature, minimal chances of replication 
interference, ease of production, and the probabil-
ity of immunization against multiple pathogens by 
a single vaccination. Their disadvantages can be the 
risk of low immunogenicity in the case of immu-
nized human hosts. Cells improperly uptake DNA 
vaccines that result in lower expression of antigens 
that’s way their success rate is low.40 Clinical trials 
on an animal are under for vectors-based vaccine 
by applying recombinant DNA technology. DNA 
vaccines have stability through activation of the in-
tracellular antigen process for adaptive immunity, 
can be easily formulated without complications as 
compared to LAVs. In the case of immunized human 
hosts, these vaccines show low immunogenicity and 
cannot produce immunity against nonstructural vi-
ral protein. The inadequate uptake by cells results in 
low antigen expression that hinders the success rate 
of DNA vaccines.91
Another DNA vaccine was reported which ex-
pressing DENV-2 prM/E fused with the immuno-
stimulatory CpG motif in order to produce protective 
immunity against the virus and to enhance the neu-
tralizing antibody response effectively as compared 
to the DENV-2 prM/E DNA vaccine.92 However, the 
DENV-2 DNA vaccine representing a recombinant 
protein carrying DENV-2 EDIII and another one 
called Escherichia maltose-binding protein (MBP) 
was utilized to immunize mice and was found to be ca-
pable to produce antibodies in mice.93 Scientists have 
evaluated the effectivenessof three nonreplicating 
DENV-2 vaccines in rhesus monkeys alone as well 
as in combination with DENV-2 prM/E DNA vaccine 
(D), DENV-2 EDIII fused with MBP recombinant 
fusion protein (R) and purified inactivated viral par-
ticles. Results have shown that alone non-replicating 
DENV-2 vaccines evoked an adequate level of anti-
bodies on the other hand combined forms of vaccine 
produced a higher level of antibodies and neutral-
ization. The titers of DP/DP/DP, DR/DR/DR and 
R/R/R vaccine were regarded as highest tigers but 
the antibody titer of D/D/D vaccine was the lowest. 
Only those animals injected with P vaccine showed 
actual protection of the immune system opposed to 
viremia.94 Some proteins specifically guiding proteins 
targeting the immune system were utilized to enhance 
the protectivity in the development of DNA vaccine. 
For example, integration of antigen sequence with ly-
sosomal membrane protein enhances the MHC class 
II antigens expression, moreover increasing the pro-
duction of CD4 T cells and anti-CD4 antigens and 
finally enhancing the DNA vaccines immunogenicity 
of the DENV-2 prM.95 A DNA vaccine can induce a 
high immune response against the targeted antigen by 
expressing an antigen that is fused with a single-chain 
Fv antibody (scFv) particularly for the DC endocytic 
receptor DEC205.94
4. Viral Vector–Based Vaccines
This approach focuses on the construction of a vi-
ral vector by inserting antigenic gene sequences that 
are capable of inducing a better antibody response. 
Some viral vectors like adenoviral vectors, Vene-
zuelan equine encephalitis virus vector, and atten-
uated measles virus vectors are designed by using 
this approach.91,96 A virus vectored dengue vaccine 
(cAdVax) was developed, consisting of bivalent 
constructs each possessing nucleotide sequences en-
coding proteins E and prM of dengue serotype 1 and 
3 together and another construct encoding the rela-
tive proteins of serotype 2 and 4. A report of NHP 
has demonstrated the production of neutralizing an-
tibodies against their specific serotypes.97,98 Based 
on the report of (cAdVax), a tetravalent vaccine 
(cAdVax-DenTV) was also developed by combin-
ing the bivalent constructs, whose protection effi-
cacy against all serotypes was praiseworthy.91
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5. Virus-Like Particle (VLP) Vaccines
These vaccines are developed by the coexpression 
of prM and E proteins of DENVs in the heterologous 
expression system of heterologous hosts.91 From the 
perspective of selecting a Virus-like Particle (VLPs) 
for vaccine development, the heterologous expres-
sion system of the yeast may be recommended 
accounted for its higher production rate and sup-
porting antigen glycosylation. A research report has 
demonstrated that we can design VLPs by express-
ing only E ectodomain proteins in yeast.99 The do-
mian III of envelop protein can also be selected for 
VLPs construction by using hepatitis B virus core 
antigens.100
VI. CONCLUSIONS
Dengue disease is the most common arboviral infec-
tious disease caused by all four serotypes of dengue 
virus, a member of Flavivirus in the Flaviriviaede 
family. Dengue virus infects millions of people 
worldwide every year. Various vector control strat-
egies are implemented to eradicate the disease, but 
the population is still at risk due to the low results of 
the said strategies. The scientific community failed 
to develop its successful vaccine due to the lack of 
understanding in immune responses against dengue 
infection. However, various dengue vaccines are in 
their developmental pipeline including LAV, inac-
tivated vaccine, recombinant subunit vaccine, viral 
vectored vaccine, and DNA vaccine, etc. A live at-
tenuated tetravalent dengue vaccine (CYD-TDV), 
trade name Dengvaxia, has completed its third clin-
ical phase, but due to lack of its potential to neu-
tralize DENV-2 and age limitation reduces its use. 
On the other hand, recombinant subunit vaccine 
can elicit the immune responses more effectively 
than the LAV, but endotoxin contamination and im-
proper protein folding also mask its efficacy. Viral 
vector-based vaccines promise their ease of genetic 
manipulation, easy detection of replication defects, 
and a satisfactory level of protein expression. DNA 
vaccines are cost-effective, genetically stable, and 
easy for mass production, but their weak immuno-
genic nature remains to be addressed. In short, in-
sights into the immune responses triggered by the 
viral components can hopefully terminate the issue 
of vaccine development.
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