potential impact of dengue virus vaccines on the brazilian ... joao_26abr1… · distinct...
TRANSCRIPT
UNIVERSIDADE DE SÃO PAULO
FACULDADE DE CIÊNCIAS FARMACÊUTICAS
Graduation on Pharmacy-Biochemistry
Potential impact of Dengue virus vaccines on the Brazilian public
health system
João Carlos Maciel Flandoli Pinheiro
Final Paper for Graduation Conclusion on
Pharmacy-Biochemistry at the School of
Pharmaceutical Sciences, University of São
Paulo.
Advisor:
Eduardo Lani Volpe da Silveira, Ph.D.
São Paulo
2017
Table of Contents
Abbreviations ........................................................................................................................ 2
1. Dengue Virus .................................................................................................................... 5
2. Epidemiology .................................................................................................................... 6
3. DENV infection, host immune response and viral evasion mechanisms ............... 9
4. Vaccine development .................................................................................................... 11
4.1 Animal Modeling and Clinical Trials ....................................................................... 12
- CYD ......................................................................................................................... 17
- DENVax .................................................................................................................. 18
- TV003 ...................................................................................................................... 19
- V180 ........................................................................................................................ 19
- D1ME100 ................................................................................................................ 20
- TDENV .................................................................................................................... 20
5. Projection considering other endemics managed with vaccination ....................... 21
- 5.1 Yellow Fever virus (YFV) ............................................................................... 22
- 5.2 Japanese Encephalitis virus ......................................................................... 22
6. Conclusion ...................................................................................................................... 23
7. References ..................................................................................................................... 24
2
Abbreviations
ANVISA Agência Nacional de Vigilância Sanitária (Regulatory Agency)
C, E, prM/M and NS Capsid, Envelope, Pre-Membrane/Membrane and Non-structural Proteins
CD Cluster of Diferentiation
CI Confidence Indicator
DENV Dengue Virus
FDA Food and Drug Administration
GMT Geometric mean titer
i.c. intracerebral
IFN Interferon
IL Interleukin
IFNAR IFNab receptors
JEV Japanese-Encephalitis Virus
LysM Lysozyme M-positive (LysM(+)) myelomonocytic cells
NESSs National Epidemiological Surveillance Systems
NHP Non-Human Primate
NIAID National Institute of Allergy and Infectious Diseases
PAHO Pan American Health Organization
PCR Polymerase chain reaction
PDK Primary Dog Kidney cells
PRRs Pattern recognition receptors
RLR RIG-I-Like receptor
RNA Ribonucleic Acid
STAT Signal Transducer and Activator of Transcription
STING Stimulator of Interferon Genes
TBEV Tick-borne encephalitis virus
TLR Toll-Like receptor
3
TNF Tumoral Necrosis Factor
VLP Virus-like Particle
VIS Gavi Vaccine Investment Strategy
WHO World Health Organization
YFV Yellow Fever Virus
ZIKV Zika Virus
4
SUMMARY
PINHEIRO, J C M F. Potential impact of Dengue virus vaccines on the Brazilian
public health system. 2017. no. f. Final Paper for Graduation Conclusion on Pharmacy-
Biochemistry at the School of Pharmaceutical Sciences, University of São Paulo, São
Paulo, 2017.
Keywords: DENGUE; VIRUS; VACCINE; BRAZIL
Dengue is an Arbovirus commonly transmitted in tropical and subtropical areas by Aedes
aegypti mosquitoes to humans. Currently, there are four circulating Dengue virus (DENV)
serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) worldwide. DENV-infected patients
may be as asymptomatic or present mild or severe manifestations. Mild symptoms refer
to fever development, headaches, myalgia and skin rash. Alterations in the vascular,
digestive, respiratory tracts and death are related to severe Dengue. Considering the
similarity between DENV infection-elicited symptoms and other arbovirus infections, the
Dengue diagnosis has been occasionally misinterpreted. In addition, flawed Brazilian
sewerage services and weak strategies to combat mosquito vector support an appropriate
scenario for the virus spread.
Several pharmaceutical industries have been working in the vaccine development to
inhibit viral transmission and disease. Those formulations have been made based on
distinct strategies, such as the use of tetravalent live attenuated, purified inactivated
viruses as well as subunit proteins. Predominantly, those vaccines are expected to
stimulate robust humoral and cellular immune responses against viral envelope (E) as
well as nonstructural (NS) proteins.
The main goal of this Paper is to review scientific bibliographic data in order to estimate
the potential impact of different vaccine candidates, already approved for use or still under
development, regarding the incidence of DENV infection. Definitively, a low cost and
effective DENV vaccine would represent a powerful tool in the control of the disease. To
enhance vaccine efficacy, improvements in vector control platforms and therapy
development should be simultaneously conducted.
5
1. Dengue Virus
Arthropod-borne viruses (arboviruses) cause several human diseases, accounting
for millions of cases each year. The majority of the ∼500 known arboviruses belong to
Bunyaviridae, Flaviviridae, Reoviridae, Rhabdoviridae, and Togaviridae viral families.
Those viruses share two major hallmarks: high mutation rates and transmission to animal
hosts through arthropods, primarily dipterans and acarines (TABACHNICK, 2016).
Flavivirus have been in the spotlights as constant threats since they have been
found to be the causative agents of some epidemics across the world. The transmission
of those single-stranded RNA enveloped viruses to humans occurs majorly through the
bite of infected bugs (Centers for Disease Control and Prevention, 2014). Dengue
(DENV), Yellow Fever (YFV), Japanese-encephalitis (JEV), St. Louis-encephalitis
(SLEV), Tick‐borne encephalitis (TBEV) and West Nile (WNV) viruses are examples of
the most common diseases caused by Flavivirus. Interestingly, Aedes mosquitoes
represent one of the most adaptable vectors capable of transmitting flavivirus infections
to humans. Besides DENV, YFV and Zika (ZIKV), these bugs can also transmit
Chikungunya virus (CHIKV), member of the Alphaviridae family, to humans
(TABACHNICK, 2016; CALISHER et al, 2016). Climate changes, deforestations,
population migration, unplanned occupation of urban areas and precarious sanitary
conditions contribute significantly to mosquito vector dissemination in tropical areas,
increasing viral transmission. Considering the lack of effective approaches to combat
these mosquitoes as well as preventative/therapeutic strategies, it explains why flavivirus
infections present that immense epidemiological impact in tropical regions worldwide
(CALISHER et al, 2016).
Among flaviviruses, DENV is one of the most rapidly emerging infections globally,
with nearly 2.5 billion people living in endemic areas, making it also the most abundant
vector-borne viral disease worldwide. Although DENV patients experience a self-limiting
febrile illness, some of them develop potentially life-threatening complications. Among
them, plasma leakage is an important characteristic of that viral infection that occasionally
can lead to systemic shock, bleeding and organ impairment. Currently, there are four
Dengue virus (DENV) serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) responsible
6
to induce that acute phase illness. Some evidences point out that most of DENV-exposed
individuals would be asymptomatic. On the other hand, DENV patients can also present
mild (fever, headaches, myalgia and skin rash) or severe symptoms (high fever, damage
to lymph and blood vessels, hemorrhage, hepatomegaly and failure of circulatory and
even neurological systems) which may progress to death. There is no specific treatment
for DENV infection. In fact, it is focused on the relief of symptoms through the
administration of painkillers and continuous hydration (Wilder-Smith et al, 2013).
Due to the possibility of absence of symptoms or development of a milder disease
upon infection, there is a concern about DENV misdiagnosis (Screaton et al., 2015) since
a great proportion of flavivirus diseases present similar clinical signs and symptoms
(Cabral-Castro et al., 2016). Within the first week upon DENV infection, the DENV
infection diagnosis relies on the detection of viral RNA or non-structural protein 1 (NS1)
through respectively quantitative RT-PCR or ELISA. DENV NS1 protein detection
presents a high sensitivity for the diagnosis of early infection, but only until day 3-4. Its
use diminishes the need of testing convalescent samples (Huang et al., 2013). Another
strategy is the use of serodiagnostic tests that detect antigen-specific antibodies in the
serum upon day 5 of infection for IgM. Rapid tests based on the detection of NS1-specific
antibodies are also available and have a reasonable window of detection during the same
stage of the disease (reviewed from Screaton et al., 2015). However, those tests are not
always available for the population of non-developed endemic countries, affecting
treatment access and inhibiting the establishment of politics to decrease viral
transmission. In order to overcome those issues, the Brazilian Ministry of Health
recommends the performance of an unspecific clinical test to predict the possibility of
developing mild or severe DENV illness. That test is called ‘Prova do Laço’ and consists
on the “arm strangulation” with a sphygmomanometer until red dots can be enumerated
in a delimited skin area. Based on the number of dots observed, the presumption is taken
(Brazilian Ministry of Health, 2016). Obviously, this test results strikes as a very
questionable and controversial diagnose method by the medical community, leaving an
important amount of cases with unclear and not certain outcomes.
2. Epidemiology
7
In the early 19th century, the Aedes aegypti was widespread all over coastal cities
of the world due to shipping vessels coming from Africa and Asia for commercial purposes.
These shipping vessels became breeding sites for the vector allowing constant contact
with humans, permitting the bug to complete their transmission cycle and spread. It
resulted in a massive transmission of DENV infection and hyperendemicity specially in
Southeast Asian countries. As numerous viral serotypes were circulating simultaneously,
the incidence of severe Dengue disease begun to be notable. In the American continent,
PAHO stimulated the use of insecticides to control YFV infection after the World War II.
That effort seemed to control also Dengue epidemics in Central and South America until
the 70s. After a long period with very low YF and Dengue transmission, the respective
governmental budgets used for that purpose started to be lowered. Consequently, DENV
infection rates scaled up during the 80s and reached skyscraper levels around 1995
(Wilder-Smith et al, 2013).
Brazil had a low incidence of DENV infections until the beginning of the 90s. In
1992, only 1.696 reported cases were reported. However, the disease incidence scaled
up considerably in the following years with 137.308 cases in 1995 and 696.472 cases in
2002. Concomitantly with that incidence enhancement, that period was marked by the
appearance of a new serotype (DENV3). Thus, that disease begun to present higher
potential to become epidemic and more severe, causing Dengue Hemorrhagic fever
(Ferreira et al., 2009). In 2002, the Brazilian government implemented the “Plano Nacional
de Controle da Dengue” (National Plan for Dengue Control) and DENV infection cases
were significantly reduced in 2003 and 2004 (Ferreira et al., 2009). Since then, the DENV
infection incidence rate is on an undeniable crescent in Brazil (Figure 1), confirming its
status of a severe public health issue (Brazilian Ministry of Health, 2016).
8
Figure 1 – Data extracted from Brazilian Ministry of Health reports
(http://portalarquivos.saude.gov.br/images/pdf/2017/fevereiro/10/Dengue-classica-ate-
2016.pdf)
Currently, WHO estimates that people from almost 130 countries are at risk of
infection with DENV. Annually, the DENV infection incidence numbers are about 400
million cases per year worldwide. In addition, it is believed that for each symptomatic
DENV infection case detected, there are three other infected asymptomatic individuals
who could still transmit the virus (WHO, 2017). The higher incidence of DENV infection,
predominately in urban cities, is due to warm weather and chaotic urbanization. Increasing
temperatures accelerates the development and physiology of Aedes aegypti, leading
mosquitoes to feed more often. Hence, as urban cities tend to be very populous and not
always have an adequate hydraulic system management with proper water provision and
garbage collection; it creates an opportunistic scenario for the disease to be spread out
more easily (Costa et al., 2016).
40
104
2 7 57
137 184
249
508
75
135
386
696
275
70
147
259 497
633
406
1.012
784
590
1.452
589
1.689
1.501
-
200
400
600
800
1.000
1.200
1.400
1.600
1.800
90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16
Rep
ort
ed
DE
NV
ca
se
s (
10
3)
in B
razil
YEAR
Historical serie of the Dengue Incidence rate in Brazil
9
3. DENV infection, host immune response and viral evasion mechanisms
DENV is an enveloped virus that packages a positive-sense (5’-3’) single-stranded
RNA that encodes for a single open reading frame (ORF). Its ORF translation results in a
polyprotein composed by three structural (capsid [C], pre-membrane/membrane [prM/M]
and envelope [E]) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and
NS5) proteins (Gack et al, 2016). During the infection, mature viral particles are
internalized by target cells, predominantly myeloid cells, including skin-resident dendritic
cells, keratinocytes, and neurons (Puccioni-Sohler et al, 2015), after interaction with
multiple types of surface receptors (reviewed by De La Guardia & Lleonart, 2014 - BioMed
Research International). They are involved in a clathrin-contained structure called
endosome. Conformational changes of E proteins culminate in a pH decrease inside the
endosomes and the fusion of viral and cellular membranes. Then, capsid protein is
released into the cell cytoplasm, where it dissociates and releases the RNA viral genome
(Valadão et al, 2016). Those viral particles migrate to endoplasmic reticulum (ER) where
they replicate into ER-derived membrane vesicles (Gack et al, 2016). For this, the viral
RNA acts utilizes the machinery of the host cell to produce polyproteins. After initiating
the RNA translation, the synthesis process switches to produce an intermediate negative-
sense (3’-5’) strand, which will be a template for the generation of multiple copies of the
positive-sense strand. Repeated cycles of translation produces multiple copies of all
structural and non-structural proteins, originating viral particles. By lowering the vesicles
pH, the viral particles are transported through the Golgi complex, being secreted out of
the cell by exocytosis (Wu et al, 2016). This replication method is graphically represented
on the Figure 2.
10
Figure 2 – The Flavivirus lifecycle (NIAID - https://www.niaid.nih.gov/research/ted-c-
pierson-phd).
Cytoplasmic replication makes these viruses vulnerable to several innate immune
sensors such as pattern recognition receptors (PRRs). RNA viral molecules are
recognized by PRRs, such as RIG-I, MDA-5 and Toll-like receptors (TLRs) 3, 7 and 8.
Adaptor molecules, such as MyD88 or TRIF, are recruited by those receptors, leading to
the nuclear translocation of transcription factors, such as c-jun/ATF2, NF-KB and
interferon-regulating factors (IRFs). In this case, those transcription factors mediate the
expression of type I interferons (IFNa/b), antiviral molecules, among other
proinflammatory cytokines that inhibit viral replication and dissemination. Interferons
induce autocrine and paracrine stress responses, such as inhibition of protein synthesis,
RNA editing and, potentially, cell death, contributing to control viral replication and
11
dissemination. Proinflammatory cytokines and chemokines, such as IL-6, IL-8, and TNF-
α, recruit and activate other cell types to the infected tissue, amplifying inflammation.
Those yielded factors are critical for the outcome of the immune response (Valadão et al,
2016).
However, DENV has developed a variety of mechanisms to evade or actively
suppress innate immune responses. These evasion strategies fall into three major
categories: sequestration or modification of viral RNA; direct inhibition of PRRs or adaptor
proteins; and antagonism of key signaling proteins downstream of PRRs. Many of the
DENV non-structural proteins are essential for the viral RNA synthesis and assembly as
well as to escape from the host immune system. For example, NS2A, NS4A and NS4B
promote INFa/b signaling inhibition, either by impairment of PRR-target interaction or
downregulation of intracellular signaling factors (Gack et al, 2016). Also, they collaborate
for the creation of intracellular membranous structures inside the cytoplasm that provides
physical protection against the host PRRs (Munoz-Jordan et al., 2003). Although the
DENV infection stimulates the IFNa/b secretion and other proinflammatory cytokines,
these mediators may also contribute to tissue lesion. Activation of cell death and oxidative
stress pathways may enhance inflammatory responses and disease severity (Valadão et
al, 2016). Whether this knowledge can be translated into the design of vaccines and
antiviral agents, it needs to be further investigated.
The rational design of live-attenuated viral strains may be possible by
systematically eliminating specific immune evasion mechanisms. A caveat of this
approach is the potential risk for reversion, but this may be overcome by disabling several
immune evasion mechanisms in combination, which may improve the safety profile of
potential vaccine strains at the expense of over-attenuation (Gack et al, 2016).
4. Vaccine development
Although it is known that strategies to combat mosquito vector have a direct impact
in reducing DENV transmission, the development of DENV protective vaccines is critical
in order to control epidemic episodes. Effective vaccines have been already available for
some flavivirus, such as YFV, JEV and TBEV. Recently, a DENV vaccine has been
12
licensed by Sanofi-Pasteur. However, its effectiveness has remained questionable in
eliciting protective immunity upon massive immunization (Roehrig et al, 2013). Another
question regarding that DENV-specific licensed vaccine is whether the generated anti-
DENV antibodies may enhance ZIKV infection in a future exposure as previously
mentioned with in vitro experiments (Pryamvada et al., 2016 - PNAS).
Multiple issues have hampered the development of a potent DENV vaccine. These
include the lack of validated animal models of disease and incomplete understanding of
immune responses following natural infection or vaccination, leading to protection or
pathogenicity and the epidemiology of four DENV serotypes (McArthur et al., 2013).
4.1 Animal Modeling and Clinical Trials
Animal models for DENV infection are needed to clarify the obscure immune-
pathological mechanisms and to test novel therapeutic and preventive approaches. There
are only 3 natural hosts for dengue; humans, non-human primates, and mosquitoes.
Although it is possible for non-human primates to be naturally infected with wild DENV,
the manifestation of clinical symptoms have been noticed only experimentally upon a high
viral load exposure (ONLAMOON et al., 2009). Currently, protection against DENV
infection have been mainly demonstrated through viremia decrease in animals. Mice have
also been approached in the development of DENV infection animal model. However,
DENV has been able to induce pathogenicity only in immunodeficient mice. STING and
STAT2 are some of the most important signaling proteins for the IFNa/b expression.
Whereas DENV NS3 and NS5 proteins efficiently degrade human STING and STAT2,
they are ineffective against murine counterparts, preventing DENV to replicate in high
titers or cause any clinical signs of disease (Pinto et al., 2015). Therefore, it is necessary
to create an animal model that presents clinical signs similar to presented by humans.
There is a variety of limitations considering DENV-challenged animal models for vaccine
candidates. Most of the models depend on unnatural and irrelevant routes of infection and
high titers of viral load. The advantages of these models relies only on the detection of
particular cell subsets and their relation with virulence mechanisms. Nonetheless, Dengue
disease is not identical to the one seen in humans. At last, the use of
13
immunocompromised mice is a factor that affects the course of disease and evaluation of
vaccines (Sarathy et al. 2015). Immunocompromised mice lacking the expression of type
I IFN receptors (IFNAR) have been proposed as an interesting model to reproduce some
aspects of Dengue seen in humans. Manifestations in IFNAR mice are the massive
production of cytokines, vascular leakage, leading to hemorrhage and non-paralytic death.
Interestingly, mice lacking IFNAR expression on either LysM+ or CD11c+ cells
(macrophages and dendritic cells respectively) remained susceptible to disease.
However, death was rare and the animals usually began to recover 5 days post infection,
same timeframe of the DENV-specific CD8+ T-cell response start. This data correlate the
viremia with the levels of type I IFNs that determine disease severity as in humans (Zust
et al. 2014).
T cells are also important against this viral infection and their magnitude were
estimated upon vaccination with a DNA vaccine based on the NS1 sequence using
BALB/c mice. Those animals were vaccinated and, then, challenged intracerebrally (i.c.)
with a mouse-brain adapted DENV. Vaccinated-challenged mice exhibited increased
frequencies of activated T cells (CD45RBlow CD4+ and CD45RBlow CD8+ T cells) in the
spleen and blood from day 5-post-infection onwards, correlating with resistance upon
DENV challenge (Figure 3). Vaccine-induced NS1-specific T cells proliferated in the liver
of DENV-challenged mice after adoptive transfer, indicating that T cells migrate to affected
peripheral organs in this animal model. Additionally, based on the reduced levels of pro-
inflammatory cytokines detected in vaccinated animals, it is suggest that the immune
response elicited by pcTPANS1 occurs in a more controlled fashion upon virus challenge
(Oliveira et al., 2016).
14
Figure 3 - Correlation between morbidity and frequency of activated T cells in spleen and
blood of DENV-challenged mice (Oliveira et al., 2016).
15
Once confirmed the efficiency of a vaccine in pre-clinical (animal model) trials, its
production is scaled up in quantity and quality control to begin clinical trials in humans.
Each phase of clinical trials have a distinct goal in the vaccine development. Phase I is
performed to demonstrate whether the vaccine can be administrated safety wise.
Whereas phase II trials evaluate the vaccine-elicited immunity, such as humoral or
adaptive responses, phase III trials represent the “test of proof” to confirm whether
vaccinees are protected against infection (Butantan Institute, 2016).
Regarding the design of clinical trials for DENV-specific vaccines, the unpredictable
nature of DENV structure, transmission and epidemics, makes it difficult, not mention
expensive. Common estimates for R&D for a single vaccine are often based on
development costs for new drugs and fall into the $1–2 billion range (Oyston et al, 2012).
As DENV exhibits a complex epidemiology with co-circulation of multiple serotypes in a
given geographic location and an unpredictable predominance of different serotypes at
different time points. Because there is no reliable way of accurately predict whether a
specific serotype is currently circulating, the determination of protective efficacy must be
considered for all serotypes simultaneously. That analysis requires multiple trial sites over
long periods of time and large numbers of volunteers (McArthur et al., 2013). In 2007,
WHO developed guidelines for the clinical evaluation of DENV vaccine in endemic
countries. It was recommended symptomatic and virologically confirmed DENV as the
primary endpoint. However, the surveillance system is unable to capture all febrile illness
related to mild dengue cases that may never result in hospitalization. Secondary
endpoints consist of efficacy evaluation against each viral serotype; efficacy after two or
more doses of vaccine; effect on the duration of hospitalization for DENV; severity of
laboratory-confirmed dengue cases; vaccine efficacy against “possible” or “probable”
DENV infection (Vannice et al, 2016).
Currently, there are some DENV vaccine candidates under distinct phases (Table
1).
16
Table 1 - Summary of Dengue Vaccines Candidates in Clinical Development (Modified
from Schwartz et al., 2015).
Vaccine
Candidate Vaccine type Main characteristics
Clinical Trial
Phase
CYD Live Attenuated
Pre-membrane and Envelope
proteins from DENV on a
Yellow Fever genome
backbone
Licensed
DENVax Live Attenuated
DENV2 attenuated via multiple
passages into primary Dog
kidney cells and mutation on
NS3 sequence
II
TV003/TV005 Live Attenuated Mutated wildtype DENV strains III
V180 Recombinant
Subunit
Wildtype Pre-membrane and
truncated Envelope proteins
through Drosophila S2 cell
expression system
I
D1ME100 DNA
DENV1 Pre-membrane and
Envelope proteins regulated by
the human cytomegalovirus
promoter/enhancer
I
TDENV Purified Inactivated
Tetravalent formulation of live
viruses propagated in Vero
cells, followed by purification
and inactivation with formalin
I
17
- CYD
CYD vaccine is a live attenuated tetravalent chimeric vaccine, based on the yellow
fever (YF) vaccine backbone. For each of the four DENV serotypes, the YF PrM and E
proteins were replaced by wildtype DENV counterparts (Schwartz et al., 2015). The main
aim of this vaccine is to stimulate naïve B cells to differentiate into antibody-secreting cells,
which would secrete protective titers of DENV-specific neutralizing antibodies (Henein et
al. 2016), however studies have shown that this specific response mainly relates to one
or few serotypes, being usually DENV4, whilst response to the others serotypes is due to
cross-reactive antibodies (Guy et al. 2015). For not having the DENV NS proteins in its
composition CYD fails to elicit optimal protective immune cellular response, as T cells will
be only exposed to epitopes from YFV NS proteins (Diamond et al. 2015). During phase
I studies conducted in naive participants, seroconversion occurred in 100% of vaccinees
to all serotypes upon 3 vaccine doses. When 2 doses were administered, 92%
seroconverted to DENV1 and 100% to the remaining serotypes (Schwartz et al., 2015).
Although local and systemic antagonistic responses were almost identical to those
recorded for other accessible live attenuated vaccines, CYD vaccination showed
correlation with increased risk of hospitalization in 2-5 year old vaccinees. Post-licensure
studies have been conducted to further assess safety concerns (WHO, 2016). Several
phase II studies have been conducted in adults and children based on 3 vaccine doses
throughout the world. In DENV-naïve population of Singapore, immunogenicity data was
yielded from 600 participants. Seroconversion was detected in 66.5% of adult vaccinees
against all viral serotypes. In immunized children, seroconversion rates were higher. Data
from another study (Peru) showed that CYD vaccination elicits higher antibody geometric
mean titer (GMT) in DENV seropositive participants at baseline than in DENV
seronegative ones. Similar data have been also observed in phase II studies conducted
in Malaysia, and Latin America (Brazil, Colombia, Honduras, Mexico, Puerto Rico)
(Schwartz et al., 2015). Two large-scale phase III efficacy trials in Latin America (Brazil,
Colombia, Honduras, Mexico, Puerto Rico) and Asia (Indonesia, Malaysia, Philippines,
Thailand, Vietnam) have been completed with more than 30,000 children and adult
participants. In both studies, participants were randomized 2:1 to vaccine or placebo and
received 3 vaccine doses. Among the 10,278 immunized children, the overall vaccine
18
efficacy was estimated in 56.5% (Asia) and 60.8% (Latin America). When CYD
vaccination was specifically evaluated for inhibition of dengue hemorrhagic fever, severe
dengue and hospitalization, it protected 88.5%, 80.8% and 67.2% of the vaccinees
respectively. Protective immunity ranged from 77.7% for DENV4 to 42.3% for DENV2 in
the Latin America study. Hence, DENV2 had the lowest degree of protection among all
viral serotypes in the Asian study. Vaccine efficacy varied by country (77.5% in Brazil to
31.3% in Mexico) as well as the dominant DENV serotype differed in those locations
(DENV4 in Brazil, DENV1 and DENV2 in Mexico). In addition, younger children presented
lower level of protection against DENV infection than older children and adults. It is likely
that limited DENV exposure and lack of immune system maturity in younger children may
reduce the vaccine ability in eliciting protection (Schwartz et al., 2015).
- DENVax
DENVax comprises a mixture of completely live-attenuated DENV2 and chimeric
DENV1, DENV3, DENV4 with attenuated DENV2 backbone. All vaccine serotypes are
based on a wild-type DENV2 isolated from a Thai symptomatic patient that was attenuated
by 53 serial passages in primary dog kidney (PDK) cells. That attenuated formulation was
named DEN-2 PDK-53 vaccine and has been tested pre-clinically and clinically since the
80s. To develop DENVax, an additional mutation in the NS3 gene was performed to
ensure attenuation (Schwartz et al., 2015). Upon a single immunization in mice,
neutralizing antibodies were generated against all serotypes, targeting their structural
proteins. The titers generated among them were indistinguishable, save for DENV4, which
elicited a considerably lower response. Also, titers of anti-NS1 antibodies, which carry
complement-fixing activity that can trigger the lysis of virus-infected cells, were generated
directly to DENV-2-NS1 and by cross-reactivity to DENV-4-NS1 proteins, were found in
the mice that were immunized by the DENV2-sepcific monovalent variation of the vaccine.
In addition, further tests with immunized animals challenged with DENV-4 NS proteins
showed a cellular response of CD4+ and CD8+ T cells predominantly producing IFN-γ
and TNFα (Fuchs et al. 2014). A phase I study was performed in order to evaluate the
safety of different routes of vaccine administration. The participant recorded, into a
memory aid, solicited systemic adverse effects (AEs) for 14 days following each
19
vaccination. Among the 80 vaccinees, systemic AEs were considered low. In terms of
immunogenicity, initial analyses showed that DENVax elicited neutralizing antibodies
against all viral serotypes (TAKEDA, 2016).
A phase II study better assessing the safety and immunogenicity of the vaccine
was conducted but results are not available yet (TAKEDA, 2016).
- TV003
TV003 vaccine is a tetravalent formulation constituted of distinct mutations for all
serotypes, increasing similarity of their kinetics. DENV1 and DENV4 present a 30-
nucleotide deletion in the 3’ untranslated region. DENV2 serotype was yielded based on
the mutated DENV4 counterpart, with replacement of PrM and E proteins. Besides the
30-nucleotide deletion in the DENV3 3’ untranslated region, that vaccine serotype has an
additional 31-nucleotide deletion upstream the deleted sequence (Schwartz et al., 2015).
The protective immunity elicited by TV003 is a combination of a robust antibody response
against all DENV serotypes and NS-specific T-cells (Durbin et al. 2013). Minor AEs,
notably mild skin rash, were seen in 63% of the vaccinees. Regarding seroconversion,
between 89%–100% of immunized participants were positive already after priming.
Seroconversion rates were not changed upon booster (DURBIN et al., 2016). In order to
estimate TV003 protection against DENV infection, vaccinees were challenged with
DENV2 with a 30-nucleotide deletion in the 3’ untranslated region 6 months after booster.
Whereas placebo recipients presented positive viremia (100%), rash (80%) and
neutropenia (20%), none of the vaccinees showed those signs of infection (Kirkpatrick et
al., 2016). TV003 has been developed by companies such as Butantan Institute,
VaBiotech, and Merck and phase III trial is ongoing in Brazil since February 2016 (Vannice
et al, 2016).
- V180
Although the development of live attenuated virus vaccines progresses from
monovalent to tetravalent trials, the interaction/interference between the viruses emerged
as an issue (Coller et al., 2011). The molecular characterization of those viral vaccine
strains and their extended dosing schedules are addressed to reduce the impact of a
20
particular virus strain interference. Moreover, the application of molecular biological tools
led to the development of several vaccine approaches (Coller et al., 2011). V180 vaccine
candidate has been developed based on the pre-membrane and truncated envelope
DENV proteins (DEN-80E) expressed by the Drosophila S2 cell expression system
(Schwartz et al., 2015). In terms of immunogenicity, pre-clinical studies were performed
in mice and macaques. They both showed that all four tested DEN-80E proteins were able
to elicit neutralizing antibodies, but in different titers. Animals were challenged with
DENV2 or DENV-4 (one animal each from each group) five months after the last
immunization. Whereas negative control animals presented positive viremia, 75% of the
immunized macaques had undetectable viremia levels (Coller et al., 2011). Although 2
Phase I trials with V180 candidate have ended, results have not been disclosed yet.
- D1ME100
D1ME100 is a plasmid DNA vector (VR1012) that contains DENV1 pre-membrane
and envelope sequences with expressions regulated by the human cytomegalovirus
promoter/enhancer. The genetic nature of the immunization proposed by this vaccine
potentially can provide long-term production of neutralizing antibodies and cellular
immune responses by mimicking the viral infectious process (Poggianella et al. 2015).
Phase I trials showed no AEs with this vaccine. Regarding its immunogenicity, the
administration of the vaccine in high dose elicited a considerable antibody response in
only 42% of vaccinees, with short duration though. In order to enhance immunogenicity,
distinct methods of DNA vaccine delivery, including different routes and ways of
administration, as well as adjuvants have been tested with this vaccine candidate
(Schwartz et al., 2015).
- TDENV
The TDENV vaccine is a Tetravalent formulation of live viruses of the strains West
Pac 74 (DENV-1), S16803 (DENV-2), CH53489 (DENV-3), and TVP360 (DENV-4)
propagated in Vero cells, purified, and inactivated with formalin. That formulation was
tested in pre-clinical trial and attested to the development of neutralizing antibody
response against all four serotypes in rhesus macaques after 2 doses. Seroconversion
21
for all four DENV serotypes was presented in 100% of the vaccinees who tolerated it well
without notable systemic or local AEs. After viral challenge, no animal presented signs of
infection, indicating that efficient neutralizing antibody response was presented against all
four DENV serotypes. However, even though merely zero cases of infection was
presented, increasing levels of RNAemia could be noticed over time (Fernandez et al,
2015). For Clinical Trials, that vaccine formulation was administrated in healthy adults to
verify its safety and humoral immunogenicity. This study is estimated to be completed by
November 2017 (U.S. Army Medical Research and Materiel Command, 2017).
5. Projection considering other endemics managed with vaccination
Considering that vaccines have shown an enormous power to decrease infectious
disease rates or even eradicate them in endemic areas, they represent one of the most
profitable long-term investments for a country. Although they rarely protect 100% of
vaccinees, very efficient vaccines have been developed against smallpox, measles,
diphtheria, tetanus, pertussis, and poliomyelitis, reducing massively numbers and costs
derived of disease incidence (and transmission), sequelae, hospitalization and mortality
over the last decades (Miller et al, 2006).
Considering that the majority of the vaccine-preventable diseases are
underreported in many countries, the estimation of disease burden is obtained through
various methodologies that consider the susceptible fraction of the population, and
calculates the natural immunity from presumed historical data of infections, immunization
coverage rates, and vaccine effectiveness. This estimation also correlates the rates of
infectivity, specific sequelae, and fatality. Another important parameter is the life
expectancy on a national level, which helps to account for causes of competing mortality,
permitting the evaluation of health outcomes as deaths, years of life lost, among others.
As any estimation model, this is based on a variety of assumptions. The more accurate
the data that supports those assumptions, closer to reality the assumption will be (Miller
et al, 2006).
Considering that other Flaviviruses have already had effective vaccines developed,
such as YF and Japanese Encephalitis viruses, it is reasonable to hypothesize that a
22
DENV protective vaccine formulation still can be made. The characteristics of those
flavivirus vaccines are:
- 5.1 Yellow Fever virus (YFV)
Yellow fever is an acute viral infection transmitted by mosquito bite, primarily the
Aedes aegypti. Common symptoms of the YFV infection are headache, nausea, joint pain,
jaundice, and myalgia. In nearly 15-20% of cases, YFV infection cause severe disease,
resulting in multiple organ failure (Miller et al, 2006). Regarding the YFV vaccine, it is a
live attenuated formulation developed with the YF17D strain, which presents a
considerable humoral response and potent induction of CD4+/CD8+ T cell response
against YF NS proteins (Barrett et al. 2009). Usually it has been administrated in 2 doses
and a booster is recommended upon 10 years of the last dose for whoever lives in or
travels to endemic regions (South America and Africa). Its AEs range from moderate
symptoms of headache, malaise, or low-grade fever to rare severe complications as
encephalitis (Miller et al, 2006). Considering its high level of protection, reaching nearly
100% of vaccinees, YFV vaccine has also been a prerequisite for those who passed
through endemic areas and are trying to entry in non-endemic countries around the world
(WHO, 2015). According to the WHO, the mass vaccination cannot eliminate YFV
because of the vast number of infected mosquitoes in urban areas of the target countries,
but it will significantly reduce viral transmission. During the 2000s, YFV vaccine was added
to the routine childhood vaccinations in most South American and African countries. While
this strategy has been considered effective on the long-term by decreasing the number of
susceptible individuals overtime, the population at-risk is not covered in the short-term.
Hundreds of YFV contaminations are yet announced every year to the World Health
Organization though (CDC).
- 5.2 Japanese Encephalitis virus
Japanese encephalitis virus (JEV) is a Flavivirus transmitted by Culex
ritaeniorhynchus mosquitoe (ITOH et al., 2016), causing encephalitis in 20-30% of
23
infected individuals and neurological sequelae in 50% of infected individuals (WHO,
2015). JEV-specific vaccine is based on high-pure inactivated JEV strain. The
implementation of that vaccine has decreased the number of annual reports of JEV
infected patients from over 1,000, during the 60s, to less than 10 in the 90s in Japan. This
dramatic reduction in the prevalence of JEV infection is the result of the introduction of a
nationwide vaccination program in 1954 with a high-purity inactivated JE vaccine.
However, humoral response to JEV vaccine seems to be short-lived as less than 20% of
the Japanese population had detectable levels of neutralizing JEV-specific antibodies,
disregarding vaccination history (Itoh et al., 2016).
6. Conclusion
Among infectious diseases, Dengue is one of the major public health problems in
Brazil. Considering the environment, social habits, and lack of efficient strategies to tackle
vector development, it is almost impossible to inhibit DENV-transmitting mosquito from
spreading (Ministério da Saúde, 2014). Currently, different approaches, other than vector
control, must be implemented in order to reduce DENV infection numbers. Also,
considering the perspective of public administration, the burden of economic
consequences of the health system and/or loss of productivity should be strongly
considered. The global economic burden of DENV infections is not well described, but it
is estimated at $2.1 billion USD each year in the Americas (Vannice et al, 2016).
Although DENV vaccine was not included in the 2013 Gavi Vaccine Investment
Strategy (VIS), a lot of progress has been done in this direction. There is already a
licensed DENV vaccine in the market and still other formulations are under development
and evaluation through clinical trials. Putting the need of safety and efficiency aside, it is
fundamental that a DENV vaccine be affordable. Most of DENV endemic areas belong to
under developing countries where wages are not high. The higher is the attendance to
vaccination against DENV, the easier the disease would be controlled, increasing the
chances for reaching the WHO goal of reducing for morbidity and mortality rates by 25%
and 50% respectively until 2020 (Vannice et al, 2016).
24
7. References
1. A McArthur, Monica; Sztein, Marcelo B; Edelman, Robert. Dengue vaccines: recent
developments, ongoing challenges and current candidates. Expert Review of
Vaccines, [s.l.], v. 12, n. 8, p.933-953, ago. 2013. Informa Healthcare.
http://dx.doi.org/10.1586/14760584.2013.815412.
2. Barrett, Alan Dt; TEUWEN, Dirk e. Yellow fever vaccine—how does it work and
why do rare cases of serious adverse events take place? Current Opinion In
Immunology, [s.l.], v. 21, n. 3, p.308-313, Jun. 2009. Elsevier BV.
http://dx.doi.org/10.1016/j.coi.2009.05.018. Available at:
<http://www.sciencedirect.com/science/article/pii/S0952791509000995>.
Accessed on Apr 20th, 2017.
3. Butantan Institute, Division of clinical development and pharmacogilance; dengue:
desafios para uma vacina 2016. Available at:
http://www.butantan.gov.br/butantan/dengue/Paginas/default.aspx Accessed on
Jan 29th, 2017.
4. Cabral-Castro, Mauro Jorge et al. Molecular and serological techniques to detect
co-circulation of DENV, ZIKV and CHIKV in suspected dengue-like syndrome
patients. Journal of Clinical Virology, [s.l.], v. 82, p.108-111, set. 2016. Elsevier BV.
http://dx.doi.org/10.1016/j.jcv.2016.07.017. Available at:
<http://www.sciencedirect.com/science/article/pii/S1386653216301871>.
Accessed on: Aug 25th, 2016.
5. Calisher, Charles H.; Woodall, John P. Yellow Fever—More a Policy and Planning
Problem than a Biological One.Emerging Infectious Diseases, [s.l.], v. 22, n. 10,
p.1859-1860, out. 2016. Centers for Disease Control and Prevention (CDC).
http://dx.doi.org/10.3201/eid2210.160875. Available at:
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038394/>. Accessed on: Oct
14th, 2016.
6. Center for Disease Control and Prevention (CDC), Viral hemorrhagic fevers
(VHFs); Available at: <http://www.cdc.gov/vhf/virus-families/flaviviridae.html>
Accessed on Nov 23rd, 2016.
25
7. Center for Disease Control and Prevention (CDC), Yellow Fever: History,
Epidemiology, and Vaccination Information; Available at: <
https://www.cdc.gov/travel-
training/local/HistoryEpidemiologyandVaccination/HistoryTimelineTranscript.pdf>
Accessed on Mar 16th, 2017.
8. Coller, Beth-ann G. et al. The development of recombinant subunit envelope-based
vaccines to protect against dengue virus induced disease. Vaccine, [s.l.], v. 29, n.
42, p.7267-7275, set. 2011. Elsevier BV.
http://dx.doi.org/10.1016/j.vaccine.2011.07.021. Available at:
<http://www.sciencedirect.com/science/article/pii/S0264410X11010425>.
Accessed on: 10 mar. 2017.
9. Costa, José Vilton; Silveira, Liciana Vaz de Arruda; Donalísio, Maria Rita. Análise
espacial de dados de contagem com excesso de zeros aplicado ao estudo da
incidência de dengue em Campinas, São Paulo, Brasil. Cadernos de Saúde
Pública, [s.l.], v. 32, n. 8, p.1-1, 2016. FapUNIFESP (SciELO).
http://dx.doi.org/10.1590/0102-311x00036915. Available at:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-
311X2016000804003&lng=pt&nrm=iso&tlng=en>. Accessed on Aug 18th. 2016.
10. Diamond, Michael s.; Pierson, Theodore c. Molecular Insight into Dengue Virus
Pathogenesis and Its Implications for Disease Control. Cell, [s.l.], v. 162, n. 3,
p.488-492, Jul, 2015. Elsevier BV. http://dx.doi.org/10.1016/j.cell.2015.07.005.
Available at: <http://www.cell.com/cell/fulltext/S0092-8674(15)00842-9>.
Accessed on Apr 25th, 2017.
11. Durbin, Anna P. et al. A 12-Month–Interval Dosing Study in Adults Indicates That
a Single Dose of the National Institute of Allergy and Infectious Diseases
Tetravalent Dengue Vaccine Induces a Robust Neutralizing Antibody Response.
Journal of Infectious Diseases, [s.l.], v. 214, n. 6, p.832-835, Feb 16th, 2016. Oxford
University Press (OUP). http://dx.doi.org/10.1093/infdis/jiw067 Available at:
https://academic.oup.com/jid/article-lookup/doi/10.1093/infdis/jiw067 Accessed
on: Apr 19th, 2017.
26
12. Durbin, A. P. et al. A Single Dose of Any of Four Different Live Attenuated
Tetravalent Dengue Vaccines Is Safe and Immunogenic in Flavivirus-naive Adults:
A Randomized, Double-blind Clinical Trial. Journal of Infectious Diseases, [s.l.], v.
207, n. 6, p.957-965, Jan 17th, 2013. Oxford University Press (OUP).
http://dx.doi.org/10.1093/infdis/jis936. Available at:
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571448/>. Accessed on: Apr
19th, 2017.
13. Fernandez, S. et al. An Adjuvanted, Tetravalent Dengue Virus Purified Inactivated
Vaccine Candidate Induces Long-Lasting and Protective Antibody Responses
Against Dengue Challenge in Rhesus Macaques. American Journal of Tropical
Medicine and Hygiene, [s.l.], v. 92, n. 4, p.698-708, Feb 2nd, 2015. American
Society of Tropical Medicine and Hygiene. http://dx.doi.org/10.4269/ajtmh.14-
0268. Available at: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385761/>.
Accessed on: Apr 13th, 2017.
14. Ferreira, Beatriz Jansen et al. Evolução histórica dos programas de prevenção e
controle da dengue no Brasil. Ciência & Saúde Coletiva, [s.l.], v. 14, n. 3, p.961-
972, jun. 2009. FapUNIFESP (SciELO). http://dx.doi.org/10.1590/s1413-
81232009000300032. Available at:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1413-
81232009000300032>. Accessed on: Dec 5th, 2016.
15. Fuchs, Jeremy et al. Investigating the efficacy of monovalent and tetravalent
dengue vaccine formulations against DENV-4 challenge in AG129
mice. Vaccine, [s.l.], v. 32, n. 48, p.6537-6543, Nov. 2014. Elsevier BV.
http://dx.doi.org/10.1016/j.vaccine.2014.08.087. Available at:
<https://www.ncbi.nlm.nih.gov/pubmed/25239488>. Accessed on: Apr 19th, 2017.
16. Gack, Michaela U; Diamond, Michael S. Innate immune escape by Dengue and
West Nile viruses. Current Opinion In Virology, [s.l.], v. 20, p.119-128, out. 2016.
Elsevier BV. http://dx.doi.org/10.1016/j.coviro.2016.09.013. Available at:
<http://www.sciencedirect.com/science/article/pii/
S1879625716301456>. Accessed on: Nov 23rd, 2016.
27
17. Guy, Bruno; Jackson, Nicholas. Dengue vaccine: hypotheses to understand CYD-
TDV-induced protection. Nature Reviews Microbiology, [s.l.], v. 14, n. 1, p.45-54,
Dec 7th, 2015. Springer Nature. http://dx.doi.org/10.1038/nrmicro.2015.2. Available
at: <http://www.nature.com/nrmicro/journal/v14/n1/full/nrmicro.2015.2.html>.
Accessed on: Apr 25th, 2017.
18. Henein, Sandra et al. Dissecting antibodies induced by a chimeric yellow fever-
dengue, live-attenuated, tetravalent dengue vaccine (CYD-TDV) in naïve and
dengue exposed individuals. Journal of Infectious Diseases, [s.l.], p.351-358, Dec
8th, 2016. Oxford University Press (OUP). http://dx.doi.org/10.1093/infdis/jiw576.
Available at: <https://academic.oup.com/jid/article-
lookup/doi/10.1093/infdis/jiw576>. Accessed on: Apr 19th, 2017.
19. Itoh, Kyoko et al. Knowledge Obtained from an Elderly Case of Japanese
Encephalitis. Intern. Med., [s.l.], v. 55, n. 17, p.2487-2490, 2016. Japanese Society
of Internal Medicine. http://dx.doi.org/10.2169/internalmedicine.55.6646. Available
at: <www.ncbi.nlm.nih.gov/pubmed/ 27580555>. Accessed on: Nov 2nd, 2016.
20. Huang, Chung-hao et al. Laboratory diagnostics of dengue fever: An emphasis on
the role of commercial dengue virus nonstructural protein 1 antigen rapid
test. Journal of Microbiology, Immunology and Infection, [s.l.], v. 46, n. 5, p.358-
365, Oct 2013. Available at: https://www.ncbi.nlm.nih.gov/pubmed/23041057.
Accessed on: Oct 23rd, 2016.
21. Khetarpal, Niyati; Khanna, Ira. Dengue Fever: Causes, Complications, and Vaccine
Strategies. Journal of Immunology Research, [s.l.], v. 2016, p.1-14, 2016. Hindawi
Publishing Corporation. http://dx.doi.org/10.1155/2016/6803098. Available at:
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971387/>. Accessed on: Aug
19th, 2016.
22. Kirkpatrick, B. D. et al. The live attenuated dengue vaccine TV003 elicits complete
protection against dengue in a human challenge model. Science Translational
Medicine, [s.l.], v. 8, n. 330, p.330-3336, Mat 16th, 2016. American Association for
the Advancement of Science (AAAS).
http://dx.doi.org/10.1126/scitranslmed.aaf1517. Available at:
<https://www.ncbi.nlm.nih.gov/pubmed/27089205>. Accessed on: Oct 25th, 2016
28
23. Miller, Mark A.; Sentz, John T. Disease and Mortality in Sub-Saharan
Africa: Chapter 12 Vaccine-Preventable Diseases. 2. ed. Washington (DC):
Jamison Dt, Feachem Rg, Makgoba Mw, Et Al., Editors. 2006. Available at:
<https://www.ncbi.nlm.nih.gov/books/NBK2284/>. Accessed on: 16 mar. 2017
24. Ministério da Saúde (BR), Secretaria de Vigilância em Saúde; Dengue Diagnostico
e Manejo Clínico Adulto e Criança, Serie A. Normas e Manuais Técnicos, v. 3,
2007. Available at:
http://bvsms.saude.gov.br/bvs/publicacoes/dengue_diagnostico_manejo_adulto_
crianca_3ed.pdf Accessed on Aug 19th, 2016
25. Ministério da Saúde (BR), Portal da Saúde – Sistema Único de Saúde; Casos de
Dengue. Brasil, Grandes Regiões e Unidades Federadas, 1990 a 2014, Updated
on 13/07/2015. Available at:
http://portalarquivos.saude.gov.br/images/pdf/2017/fevereiro/10/Dengue-classica-
ate-2016.pdf Accessed on Mar 19th, 2016
26. Ministério da Saúde (BR), Portal da Saúde – Combate à dengue, Updated on
28/07/2014. Available at: http://www.brasil.gov.br/saude/2009/11/combate-a-
dengue Accessed on Dec 08th, 2016
27. Ministério da Saúde (BR), Secretaria de Vigilância em Saúde; Monitoramento dos
casos de dengue, febre de
Chikungunya e febre pelo vírus Zika até a Semana Epidemiológica 27, 2016.
Available at: <http://portalsaude.saude.gov.br/images/pdf/2016/agosto/10/2016-
026--2.pdf>. Accessed on Aug 19th, 2016
28. Munoz-Jordan, J. L. et al. Inhibition of interferon signaling by dengue virus.
Proceedings of the National Academy of Sciences, [s.l.], v. 100, n. 24, p.14333-
14338, Nov 11th. 2003. Proceedings of the National Academy of Sciences.
http://dx.doi.org/10.1073/pnas.2335168100. Available at:
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC283592/>. Accessed on: Apr 3rd,
2017.
29. Nunes, Juliana Dengue: Etiologia, patogênese e suas implicações a nível global.
Dissertação para obtenção do Grau de Mestre em: Medicina Available at:
29
<www.fcsaude.ubi.pt/thesis2/anexo.php?id=81bffdd4d1e0b6c0>. Accessed on
Dec 12th, 2016
30. Oliveira, Edson R. A. et al. Aspects of T Cell-Mediated Immunity Induced in Mice
by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the
Intracerebral Route. Plos One, [s.l.], v. 11, n. 9, e0163240, 15 set. 2016. Public
Library of Science (PLoS). http://dx.doi.org/10.1371/journal.pone.0163240.
Available at: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024998/>.
Accessed on: Jan 16th, 2017.
31. Onlamoon, N. et al. Dengue virus-induced hemorrhage in a nonhuman primate
model. Blood, [s.l.], v. 115, n. 9, p.1823-1834, Dec 30th, 2009. American Society of
Hematology. http://dx.doi.org/10.1182/blood-2009-09-242990. Disponível em:
<https://www.ncbi.nlm.nih.gov/pubmed/20042723>. Accessed on em: Mar 25th,
2017.
32. Oyston, P.; Robinson, K. The current challenges for vaccine development. Journal
of Medical Microbiology, [s.l.], v. 61, n. 7, p.889-894, Feb 9th, 2012. Microbiology
Society. http://dx.doi.org/10.1099/jmm.0.039180-0. Available at:
<http://jmm.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.039180
-0#tab2>. Accessed on: Jan 30th, 2017
33. Pinto, Amelia K. et al. Defining New Therapeutics Using a More Immunocompetent
Mouse Model of Antibody-Enhanced Dengue Virus Infection. Mbio, [s.l.], v. 6, n. 5,
p.13-15, 15 set. 2015. American Society for Microbiology.
http://dx.doi.org/10.1128/mbio.01316-15. Available at:
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600115/>. Accessed on: Jan
22nd, 2017.
34. Poggianella, Monica et al. Dengue E Protein Domain III-Based DNA Immunization
Induces Strong Antibody Responses to All Four Viral Serotypes. Plos Neglected
Tropical Diseases, [s.l.], v. 9, n. 7, Jul 28th, 2015. Public Library of Science (PLoS).
http://dx.doi.org/10.1371/journal.pntd.0003947. Available at:
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517776/>. Accessed on: Apr
20th, 2017.
30
35. Puccioni-Sohler, Marzia; Rosadas, Carolina. Advances and new insights in the
neuropathogenesis of dengue infection. Arquivos de Neuropsiquiatria, [s.l.], v. 73,
n. 8, p.698-703, ago. 2015. FapUNIFESP (SciELO).
http://dx.doi.org/10.1590/0004-282x20150074. Available at:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0004-
282X2015000800698>. Accessed on: 02 abr. 2017.
36. Roehrig, John T; Barrett, Alan Dt. Flavivirus Infections in Humans. Els, [s.l.], p.47-
58, 13 jun. 2013. Wiley-Blackwell.
http://dx.doi.org/10.1002/9780470015902.a0002233.pub3. Available at:
<http://www.els.net/WileyCDA/ElsArticle/refId-a0002233.html>. Accessed on: Nov
23rd, 2016.
37. Sarathy, Vanessa V. et al. Mouse models of dengue virus infection for vaccine
testing. Vaccine, [s.l.], v. 33, n. 50, p.7051-7060, Dec 2015. Elsevier BV.
http://dx.doi.org/10.1016/j.vaccine.2015.09.112. Available at:
<http://www.sciencedirect.com/science/article/pii/S0042682209006461>.
Accessed on: Apr 16th, 2017.
38. Schwartz, Lauren M. et al. The dengue vaccine pipeline: Implications for the future
of dengue control. Vaccine, [s.l.], v. 33, n. 29, p.3293-3298, jun. 2015. Elsevier BV.
http://dx.doi.org/10.1016/j.vaccine.2015.05.010. Available at:
<http://www.sciencedirect.com/science/article/pii/S0264410X15006313>.
Accessed on: Jan 23rd 2017
39. Screaton, Gavin et al. New insights into the immunopathology and control of
dengue virus infection. Nat Rev Immunol, [s.l.], v. 15, n. 12, p.745-759, 25 Nov.
2015. Nature Publishing Group. http://dx.doi.org/10.1038/nri3916. Available at:
<http://www.nature.com/nri/journal/v15/n12/full/nri3916.html>. Accessed on
Aug16th, 2016.
40. Takeda (Org.). Study to Investigate the Safety and Immunogenicity of a Tetravalent
Chimeric Dengue Vaccine in Healthy Volunteers Between the Ages of 1.5 - 45
Years in: ClinicalTrials.gov. 2016. Available at:
<https://clinicaltrials.gov/ct2/show/NCT01511250?term=DENVax&rank=4&submit
;_fld_opt=>. Accessed on: Jan 29th, 2017.
31
41. Tabachnick, Walter J. Climate Change and the Arboviruses: Lessons from the
Evolution of the Dengue and Yellow Fever Viruses. Annual Review of Virology,
[s.l.], v. 3, n. 1, p.125-145, Sep 29th, 2016. Annual Reviews.
http://dx.doi.org/10.1146/annurev-virology-110615-035630. Available at:
<http://www.annualreviews.org/doi/full/10.1146/annurev-virology-110615-
035630?url_ver=Z39.88-
2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed&>. Accessed on: Oct
11th, 2016
42. U.S. Army Medical Research and Materiel Command (Org.). A Two-dose Primary
Vaccination Study of a Tetravalent Dengue Virus Purified Inactivated Vaccine vs.
Placebo in Healthy Adults In: ClinicalTrials.gov. 2017. Available at:
<https://clinicaltrials.gov/ct2/show/record/NCT01666652?term=TDENV&rank=5>.
Accessed on: Apr 12th, 2017.
43. Valadão, Ana L. C.; Aguiar, Renato S.; Arruda, Luciana B. de. Interplay between
Inflammation and Cellular Stress Triggered by Flaviviridae Viruses. Frontiers in
Microbiology, [s.l.], v. 7, Aug 25th, 2016. Frontiers Media SA.
http://dx.doi.org/10.3389/fmicb.2016.01233. Available at:
<http://journal.frontiersin.org/article/10.3389/fmicb.2016.01233/full>. Accessed on:
Dec 19th, 2016
44. Vannice, Kirsten S.; Durbin, Anna; Hombach, Joachim. Status of vaccine research
and development of vaccines for dengue. Vaccine, [s.l.], v. 34, n. 26, p.2934-2938,
jun. 2016. Elsevier BV. http://dx.doi.org/10.1016/j.vaccine.2015.12.073. Available
at: <http://www.sciencedirect.com/science/article/pii/S0264410X16002930>.
Accessed on: Mar 18th, 2017.
45. Wilder-Smith, Annelies; Murray; Quam, Mikkel. Epidemiology of dengue: past,
present and future prospects. Clinical Epidemiology, [s.l.], p.299-309, Aug 2013.
Dove Medical Press Ltd.. http://dx.doi.org/10.2147/clep.s34440. Available at:
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753061/>. Accessed on: Mar
30th, 2017.