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

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