CHAPTER SEVEN Dengue Virus Vaccine Development Lauren E. Yauch, Sujan Shresta 1 Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA 1 Corresponding author: e-mail address: [email protected]Contents 1. Virology and Epidemiology of DENV Infection 316 2. Adaptive Immune Response to DENV 317 3. Dengue Vaccine Objectives and Challenges 320 4. Animal Models for Testing Dengue Vaccine Candidates 323 5. Dengue Vaccine Approaches 325 5.1 Recombinant subunit protein vaccines/subviral particles 325 6. DNA Vaccines 329 7. Viral Vectored Vaccines 333 7.1 Vaccinia 333 7.2 Adenovirus vectors 334 7.3 Alphavirus replicon particles 335 8. Inactivated Whole Virus 336 9. Live Attenuated 339 9.1 University of Hawaii/WRAIR 340 9.2 Mahidol University 343 9.3 CDC/Inviragen 345 9.4 NIAID/NIH 346 9.5 DENV Chimeras 350 9.6 Acambis/Sanofi Pasteur (ChimeriVax) 351 10. Moving Forward 355 References 357 Abstract Dengue virus (DENV) is a significant cause of morbidity and mortality in tropical and subtropical regions, causing hundreds of millions of infections each year. Infections range from asymptomatic to a self-limited febrile illness, dengue fever (DF), to the life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The expanding of the habitat of DENV-transmitting mosquitoes has resulted in dramatic increases in the number of cases over the past 50 years, and recent outbreaks have occurred in the United States. Developing a dengue vaccine is a global health priority. DENV vaccine development is challenging due to the existence of four serotypes of the Advances in Virus Research, Volume 88 # 2014 Elsevier Inc. ISSN 0065-3527 All rights reserved. http://dx.doi.org/10.1016/B978-0-12-800098-4.00007-6 315
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CHAPTER SEVEN
Dengue Virus VaccineDevelopmentLauren E. Yauch, Sujan Shresta1Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA1Corresponding author: e-mail address: [email protected]
Contents
1.
AdvaISSNhttp:/
Virology and Epidemiology of DENV Infection
nces in Virus Research, Volume 88 # 2014 Elsevier Inc.0065-3527 All rights reserved./dx.doi.org/10.1016/B978-0-12-800098-4.00007-6
316
2. Adaptive Immune Response to DENV 317 3. Dengue Vaccine Objectives and Challenges 320 4. Animal Models for Testing Dengue Vaccine Candidates 323 5. Dengue Vaccine Approaches 325
5.1
Recombinant subunit protein vaccines/subviral particles 325 6. DNA Vaccines 329 7. Viral Vectored Vaccines 333
Inactivated Whole Virus 336 9. Live Attenuated 339
9.1
University of Hawaii/WRAIR 340 9.2 Mahidol University 343 9.3 CDC/Inviragen 345 9.4 NIAID/NIH 346 9.5 DENV Chimeras 350 9.6 Acambis/Sanofi Pasteur (ChimeriVax) 351
10.
Moving Forward 355 References 357
Abstract
Dengue virus (DENV) is a significant cause of morbidity and mortality in tropical andsubtropical regions, causing hundreds of millions of infections each year. Infectionsrange from asymptomatic to a self-limited febrile illness, dengue fever (DF), to thelife-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Theexpanding of the habitat of DENV-transmitting mosquitoes has resulted in dramaticincreases in the number of cases over the past 50 years, and recent outbreaks haveoccurred in the United States. Developing a dengue vaccine is a global health priority.DENV vaccine development is challenging due to the existence of four serotypes of the
virus (DENV1–4), which a vaccine must protect against. Additionally, the adaptiveimmune response to DENV may be both protective and pathogenic upon subsequentinfection, and the precise features of protective versus pathogenic immune responsesto DENV are unknown, complicating vaccine development. Numerous vaccinecandidates, including live attenuated, inactivated, recombinant subunit, DNA, and viralvectored vaccines, are in various stages of clinical development, from preclinical tophase 3. This review will discuss the adaptive immune response to DENV, dengue vac-cine challenges, animal models used to test dengue vaccine candidates, and historicaland current dengue vaccine approaches.
1. VIROLOGY AND EPIDEMIOLOGY OF DENV INFECTION
Dengue virus (DENV) is the etiologic agent of dengue fever (DF), the
most prevalent arthropod-borne viral illness in humans. DENV belongs to
the Flaviviridae family and is related yellow fever virus (YFV), hepatitis
C virus, West Nile virus, Japanese encephalitis virus (JEV), and St. Louis
encephalitis virus. DENV is an enveloped virus with a single-stranded,
positive-sense RNA genome. The DENV genome is 10.7 kb and contains
a 50methyl guanosine cap, 50untranslated region (UTR), single open reading
frame, and a 30UTR (Clyde, Kyle, & Harris, 2006). The RNA genome is
translated as a single polyprotein that is then cleaved into three structural
proteins (capsid (C), premembrane (prM), and envelope (E)) and seven non-
Bunyaratvej, 1987). Five JEV- and DENV-naive volunteers and five
JEV-immune volunteers were vaccinated. One patient became viremic,
and all developed neutralizing antibodies that lasted for 1.5 years.
344 Lauren E. Yauch and Sujan Shresta
DENV2-specific CD4þ and CD8þ T-cell responses were detected in all
vaccinees (Dharakul et al., 1994).When given in a bivalent formulation with
a DENV4 vaccine strain, 1036 PDK 48, all subjects developed neutralizing
antibodies against DENV2 and DENV4 (Bhamarapravati & Yoksan, 1989).
The 16681-PDK-53 vaccine was also found to be safe and immunogenic in
10 flavivirus-naive American volunteers, who developed a DENV2 neutral-
izing antibody response that lasted for 2 years (Vaughn et al., 1996).
Vaccine strains from each serotype obtained by passage through PDK
cells or primary GMK cells were selected and tested in monovalent, bivalent,
trivalent, and tetravalent vaccinations in Thai adults (Bhamarapravati &
Sutee, 2000). The strains used were DENV1 PDK-13, DENV2 PDK-53,
DENV3 PGMK-30/F3, and DENV4 PDK-48. The vaccine was safe and
did not induce clinically significant symptoms. Of the volunteers that
seroconverted, most had neutralizing antibodies 2 years after monovalent
vaccination. All bivalent and trivalent vaccine recipients seroconverted to
all serotypes in the vaccine, and of the tetravalent recipients, four of six
developed neutralizing antibodies to all four serotypes, whereas two
seroconverted to DENV1, 2, and 3 but not DENV4.
The vaccine strains were produced by Aventis Pasteur and tested in a
phase 1 trial in the United States in 40 flavivirus-naive adults (Kanesa-
thasan et al., 2001). Subjects received a single dose of a monovalent vaccine
or the tetravalent vaccine (containing 3.47–3.9 log10 PFU of each serotype).
Mild symptoms including fever, headache, malaise, rash, and transient neu-
tropenia were observed in the monovalent recipients. Tetravalent vaccina-
tion was more reactogenic than monovalent vaccination, and one volunteer
developed a dengue-like syndrome. Viremia was detected in DENV3 and
DENV4monovalent recipients, and DENV3was detected in the tetravalent
vaccine recipients. All of the of DENV2, 3, and 4 monovalent recipients but
only 60% of the DENV1 recipients seroconverted. Of the tetravalent recip-
ients, only one of ten seroconverted to all four serotypes, and neutralizing
antibody responses were directed primarily to DENV3. The vaccine
induced DENV-specific T-cell responses (as measured by in vitro prolifera-
tion, IFN-g production, and cytotoxicity) in the tetravalent vaccine recip-
ients; however, the responses to the four serotypes were not equivalent
(Rothman et al., 2001).
In an attempt to achieve a more balanced antibody response, seven tet-
ravalent vaccine formulations were tested that differed in overall viral dose
and the dose of each serotype (Sabchareon et al., 2002). Fifty-nine flavivirus-
naive Thai adults received two vaccine doses 6 months apart. Five volunteers
345Dengue Vaccines
developed a DF-like illness, with headache, fever, and myalgia the most
common symptoms. Some hematologic abnormalities were also observed
including decreases in platelets, neutrophils, and lymphocytes, and some
subjects had increased AST and ALT levels. The second dose was less
reactogenic, but viremia was detected after both doses. After the second
dose, 76% of subjects seroconverted to three serotypes and 71%
seroconverted to all four. The DENV3 component was dominant; viremia
detected after the first dose was mainly DENV3, all subjects seroconverted to
DENV3 after one dose, and neutralizing antibody titers were highest
against DENV3.
Two formulations of a tetravalent vaccine that contained less DENV3
than previous formulations were then tested in Thai children (Sabchareon
et al., 2004). Children 5–12 years of age received three immunizations—
the second was given 3–5 months after first, and the third was given 8–12
months after the second. The vaccines were moderately reactogenic and
induced symptoms including fever, myalgia, and rash. There were five
severe reactions including one DF-like illness. After three doses, 89% and
100% of the recipients seroconverted to all four serotypes. DENV3 was still
dominant, as indicated by a high prevalence of DENV3 viremia and high
neutralizing antibody titers against DENV3.
A planned phase 1b trial to test two formulations of the vaccine in adult
Caucasians in Australia was halted after 10 recipients received one dose and
developed a mild DF-like syndrome due to the DENV3 component
(Kitchener et al., 2006). In an attempt to attenuate DENV3, the vaccine
strain was plaque-purified and adapted to Vero cells (Sanchez et al.,
2006). The Vero-adapted dengue serotype 3 vaccine, VDV3, was attenuated
in vitro and in monkeys and was next tested in 15 volunteers in Hong Kong.
All subjects had adverse reactions and the trial was halted. As a balanced
immune response was not achieved with these vaccine candidates, they were
not pursued further.
9.3. CDC/InviragenAnother live attenuated candidate was developed at the CDC and has been
licensed by Inviragen. Chimeric viruses were cloned with the DENV2
PDK-53 vaccine strain developed at Mahidol University as a backbone,
and the DENV2 structural proteins were replaced with the structural pro-
teins from DENV1, 3, or 4 to create the tetravalent vaccine, DENVax.
Attenuating mutations in PDK-53 are outside of the structural genes
346 Lauren E. Yauch and Sujan Shresta
(Butrapet et al., 2000); therefore, all four chimeric strains should retain the
DENV2 PDK-53 attenuation markers. DENV2/DENV1 chimeras were
created using the C, M, and E proteins of the Mahidol DENV1 PDK-13
vaccine virus or wild-type DENV1 16007 and were found to be attenuated
in vitro and in mice (Huang et al., 2000).
DENV2/1, DENV2/3, and DENV2/4 chimeras were created by clon-
ing prM and E from wild-type DENV1 (strain 16007), DENV3 (strain
16562), and DENV4 (strain 1036) into two genetic variants of the DENV2
PDK-53 vaccine virus, or the parental strain, 16681 (Huang et al., 2003).
The chimeras retained the DENV2 PDK-53 attenuation markers, including
temperature sensitivity, small plaque size in LLC-MK2 cells, lack of neu-
rovirulence in newborn mice, and reduced replication in C6/36 mosquito
cells. Monovalent and tetravalent chimeric vaccine (DENVax) formulations
were tested in AG129 mice (Brewoo et al., 2012; Huang et al., 2003).
Monovalent DENVax-1, 2, or 3 significantly protected against lethal
DENV1 or DENV2 challenge. Tetravalent vaccination induced neutraliz-
ing antibody responses against all four serotypes and protected against chal-
lenge with DENV1 or DENV2.
Three different formulations, differing in the dose of each serotype, of
the tetravalent chimeric DENVax vaccine were tested in cynomolgus
macaques (Osorio, Brewoo, et al., 2011). Monkeys were given two vacci-
nations 60 days apart. Low-level DENV2 viremia was detected, yet all mon-
keys developed neutralizing antibodies against all four serotypes after one or
two doses. Monkeys also developed a DENV2-specific T-cell response. The
most balanced antibody response was observed with the formulation con-
taining 103 PFU of DENV1 and DENV2 and 105 PFU of DENV3 and
DENV4. All monkeys were completely protected against challenge with
DENV3 or DENV4 30 days after the second immunization, and the
high-dose formulation (105 PFU of each serotype) completely protected
against DENV1 and DENV2 as well. Based on these results, tetravalent
DENVax is being tested in phase 1 clinical trials (Osorio, Huang,
Kinney, & Stinchcomb, 2011), and a phase 2 study in healthy volunteers
between 1.5 and 45 years of age began in 2011 (Clinicaltrials.gov
NCT01511250).
9.4. NIAID/NIHA genetics approach was undertaken by researchers in the Laboratory of
Infectious Diseases at the National Institute of Allergy and Infectious
347Dengue Vaccines
Diseases (NIAID) with the goal of attenuating the virus without significantly
reducing immunogenicity. Reverse genetics was used to introduce dele-
tions, from 30 to 262 nucleotides (nt), into the 30UTR of DENV4 cDNA
(Men, Bray, Clark, Chanock, & Lai, 1996). Mutants that were attenuated in
LLC-MK2 cells were selected and tested in rhesus monkeys. Some mutants
were attenuated in vivo, in terms of reduced viremia and neutralizing anti-
body titers, compared with the parental wild-type DENV4 virus. ADENV4
30 nt 30UTR deletion mutant (rDENV4D30) that was attenuated in mon-
keys was selected and tested in 20 healthy adults in a phase 1 trial (Durbin
et al., 2001). Volunteers received 105 PFU s.c. Low titer viremia was
detected in 14 volunteers, and 100% developed neutralizing antibody
responses against DENV4. The vaccine was well tolerated: Asymptomatic
rash was observed in subjects with viremia, and 5 volunteers had a transient
increase in serum ALT levels. The vaccine was attenuated for mosquitoes as
well. Compared with the wild-type parental virus, the vaccine strain was
restricted in infecting A. aegypti midgut and in disseminating from the mid-
gut to the salivary gland. In addition, vaccine recipients did not transmit the
virus to A. albopictus mosquitoes (Troyer et al., 2001).
The rDENV4D30 vaccine was further evaluated in phase 2 placebo-
controlled trial (Durbin et al., 2005). A dose deescalation was done, and vac-
cinees (20 per group) received 103, 102, or 101 PFU. All doses were well
tolerated and immunogenic. Some recipients developed a mild rash and
neutropenia, but only 1/60 had an elevated serum ALT level. Almost all
recipients (97%) seroconverted (defined as a � fourfold increase in neutral-
izing antibody titers) to DENV4 after a single inoculation. These results
supported the inclusion of this vaccine strain in a tetravalent formulation.
In parallel, DENV4 mutants were generated in an attempt to derive a
vaccine candidate that would not induce the hepatotoxicity observed in vol-
unteers receiving 105 PFU of the rDENV4D30 vaccine (Hanley, Lee,
Blaney, Murphy, & Whitehead, 2002). Five attenuating mutations were
introduced into rDENV4D30 and were tested in SCID-HuH-7 mice and
rhesus monkeys (Hanley et al., 2004). One mutant (rDENV4D30-200,201) that was significantly attenuated in rhesus monkeys compared with
wild-type DENV4 and rDENV4D30 was selected and tested in a phase 1
trial (McArthur et al., 2008). Volunteers received 105 PFU of
rDENV4D30-200,201, which was well tolerated; no ALT elevations or
viremia were detected, and all 20 volunteers seroconverted after one dose.
Toward the goal of creating a tetravalent vaccine, the group introduced
the 30 nt 30UTR deletion into a full-length DENV1 cDNA clone to create
348 Lauren E. Yauch and Sujan Shresta
rDENV1D30 (Whitehead, Falgout, et al., 2003). This virus was attenuated
similarly to rDENV4D30 in rhesus monkeys and completely protected
against DENV1 challenge, with no viremia detected in vaccinated monkeys.
A phase 1 study of the rDENV1D30 DENV1 vaccine was conducted in
adult volunteers (Durbin et al., 2006a). Twenty vaccinees received 103
PFU, which was well tolerated. The most common adverse events were
an asymptomatic rash and neutropenia, which were observed in 40% and
45% of the recipients, respectively. Viremia was detected in 9/20 subjects
and was slightly higher titer than rDENV4D30-induced viremia. The vac-
cine was highly immunogenic, as 95% of the recipients seroconverted and
had neutralizing antibodies against DENV1 that lasted for the 6 months of
the study. A subsequent study found a second immunization with
rDENV1D30 4 or 6 months after the first dose was safe; however, it was
not infectious and it did not boost antibody titers, indicating the first vacci-
nation induced sterilizing immunity that lasted for at least 6 months (Durbin,
Whitehead, et al., 2011).
For DENV3, unlike DENV1 and DENV4, the D30 mutation was not
sufficiently attenuating. rDENV3D30 was not attenuated in mosquitoes,
SCID-HuH-7 mice, or monkeys (Blaney, Hanson, Firestone, et al.,
2004). As an alternate attenuating strategy, the DENV3 M and E proteins
were cloned into the rDENV4 backbone to create rDENV3/4(ME) and
rDENV3/4D30(ME) chimeras, which were attenuated in mice, mosqui-
toes, and rhesus monkeys. The two chimeras were comparably attenuated,
indicating the D30 mutation did not confer additional attenuation. No vire-
mia was detected in immunized monkeys yet all seroconverted, and they
were protected against challenge with the parental DENV3.
Additional DENV3 vaccine candidates were created, including
rDENV3D30/31, which contains an additional 31 nt deletion in the
30UTR, and rDENV3-30D4D30, which was created by replacing the entire30UTR of rDENV3 with the 30UTR of rDENV4D30 (Blaney et al., 2008).Both viruses were attenuated in SCID-HuH-7 mice and rhesus monkeys;
immunization of monkeys resulted in neutralizing antibody responses and
protection from wild-type DENV3 challenge. rDENV3D30/31 was also
attenuated for mosquitoes.
Similarly, the D30 mutation in DENV2 did not sufficiently attenuate the
virus to be considered for a human vaccine. rDENV2D30 was attenuated inSCID-HuH-7 mice and not infectious for A. aegypti mosquitoes, but was
only slightly attenuated in rhesus monkeys compared with rDENV2 and
wild-type DENV2 (Blaney, Hanson, Hanley, et al., 2004). To further
349Dengue Vaccines
attenuate rDENV2D30, a point mutation in NS3 that had been previously
demonstrated to attenuate rDENV4D30 (Hanley et al., 2004) was made.
rDENV2D30-4995 was found to be further attenuated in SCID-HuH-7
mice compared with rDENV2D30. In other approaches to create DENV2
vaccine candidates, the structural genes (CME or ME) of DENV2 were
cloned into rDENV4 or rDENV4D30 (Whitehead, Hanley, et al., 2003).
Chimeras (without the D30 deletion) were attenuated in SCID-HuH-7
mice, mosquitoes, and rhesus monkeys. rDENV2/4D30(CME) was more
attenuated than rDENV2/4(CME) and did not replicate in monkeys;
rDENV2/4(ME) was similarly attenuated when cloned with or without
the D30 deletion.
Due to its attenuation and immunogenicity, rDENV2/4D30(ME) was
deemed a promising vaccine candidate and was tested in 20 DENV-naive
adults (Durbin et al., 2006b). The volunteers received 103 PFU, which
was safe and immunogenic. A mild asymptomatic rash and mild neutropenia
were observed in some subjects. All volunteers seroconverted to DENV2
and neutralizing antibodies were maintained for the 6 months of the study.
Low magnitude viremia was detected in 11 volunteers, and the D30 muta-
tion was unchanged in the viremic volunteers, confirming that the mutation
was stable.
Three tetravalent vaccine formulations were tested in animals (Blaney
et al., 2005). TV-1 was composed of 105 PFU of the four D30 viruses;
TV-2 contained 105 PFU of rDENV1D30, rDENV4D30, rDENV2/
4D30, and rDENV3/4D30; and TV-3 contained 105 PFU of rDENV1D30,rDENV2D30, and rDENV4D30, and 106 PFU of rDENV3/4D30. TV-1and TV-2 were attenuated in SCID-HuH-7mice, and all three formulations
were attenuated in rhesus monkeys. TV-1- and TV-3-immunized monkeys
all seroconverted after one dose, whereas TV-2 required a booster immu-
nization to achieve high titers against DENV2 and DENV3. Boosting at
4 months, but not 1 month, increased neutralizing antibody titers.
A single dose of TV-2 protected against challenge with DENV1, 3, and
4, and two doses protected from challenge with DENV2. Two doses of
TV-3 also completely protected against DENV2 challenge. These results
supported testing TV-2 and TV-3 in clinical trials.
A phase 1 trial investigated a single dose of four different formulations of
a live tetravalent vaccine in 113 flavivirus-naive volunteers (Durbin et al.,
2013). The vaccines were well tolerated, with no SAE or fever induced
in any subject. The only side effect that occurred with a significantly higher
incidence in vaccinees compared with placebo recipients was an
350 Lauren E. Yauch and Sujan Shresta
asymptomatic rash observed in 64.2% of vaccinees. Low-level viremia was
detected in most (73%) recipients, and in the majority (64%) of viremic sub-
jects, one serotype of virus was detected. One dose of each formulation
induced a trivalent or better neutralizing antibody response in 75–90% of
the volunteers. Black race correlated with lower seropositivity and a reduced
incidence of viremia, which was interesting as the black race is associated
with resistance to DENV infection (Blanton et al., 2008; Halstead et al.,
2001). Formulation TV003, containing 103 PFU each of rDENV1D30,rDENV2/4D30, rDENV3D30/31, and rDENV4D30, induced the most
balanced neutralizing antibody response and a trivalent or better response
in 90% of recipients after a single dose. However, only 50% of recipients
seroconverted to DENV2. Phase 1 trials testing two different formulations
(TV003 and TV005, which contains a higher dose of rDENV2/4D30 than
TV003) of the tetravalent vaccine (TetraVax-DV) began in 2011 in
flavivirus-naive adults (Clinicaltrials.gov NCT01436422) and flavivirus-
immune adults (NCT01506570). A phase 2 trial in Brazil is planned.
The safety and immunogenicity of vaccination of DENV-immune indi-
viduals was investigated (Durbin, Schmidt, et al., 2011). Individuals who
had received a monovalent DENV vaccine were given a second immuniza-
tion with a heterotypic monovalent attenuated vaccine 0.6–7.4 years later.
Replication and safety were comparable in immunized and naive volunteers.
In contrast to naive individuals, most volunteers who received a second
DENV vaccination developed a broad, heterotypic neutralizing antibody
response. However, in one cohort, preexisting DENV2 immunity impaired
seroconversion to a DENV1 vaccine.
The D30 vaccines have a number of advantages. Attenuation is due to
deletions in 30UTR, so both T-cell and antibody responses can be induced
against wild-type DENV structural and nonstructural proteins. Deletion
mutants are more stable than point mutations and therefore these strains
are unlikely to revert to wild-type viruses. In addition, as the four vaccine
strains contain the same deletion, potential recombination between the four
viruses will not lead to reversion of wild-type virus.
9.5. DENV ChimerasChimeric viruses were constructed using recombinant DNA technology
(Bray & Lai, 1991). Using the cDNA of DENV4, the C, prM, and
E genes were replaced with structural genes from DENV1 or DENV2.
The DENV2/DENV4 chimera was attenuated, providing a proof of
351Dengue Vaccines
concept for producing attenuated, chimeric dengue vaccine strains. The
chimeras were attenuated in rhesus monkeys (Bray, Men, & Lai, 1996).
Monkeys vaccinated with DENV1/DENV4 or DENV2/DENV4 chimeras
developed neutralizing antibodies against DENV1 and DENV2, respec-
tively, and were protected against challenge with DENV1 or DENV2.
Monkeys immunized with an equal mixture of DENV1/DENV4 and
DENV2/DENV4 chimeras were protected from challenge with DENV1
or DENV2.
9.6. Acambis/Sanofi Pasteur (ChimeriVax)Research begun at the NIH and St. Louis University (Bray & Lai, 1991;
Chambers, Nestorowicz, Mason, & Rice, 1999) and continued at Acambis
(now part of Sanofi Pasteur) resulted in the creation of chimeric viruses con-
taining the DENV structural proteins on the YF 17D backbone. The YF
17D vaccine backbone was selected because of the safety, long duration
of immunity, and rapid onset of immunity induced by the YFV 17D vac-
cine, which has been used for over 60 years. To create a DENV2 chimeric
strain, ChimeriVax-DENV2, the prM and E genes from the DENV2 PUO-
218 strain were cloned into a cDNA infectious clone of 17D (Guirakhoo
et al., 2000). ChimeriVax-DENV2 was nonneurovirulent for 4-week-old
mice and was genetically stable. Inoculation of rhesus monkeys resulted
in brief viremia, a neutralizing antibody response, and complete protection
from challenge with wild-type DENV2. DENV1, DENV3, and DENV4
chimeras were then constructed using the prM/E sequences from DENV
clinical isolates (Guirakhoo et al., 2001). The chimeras replicated to high
titers in Vero cells, were nonneurovirulent in 4-week-old mice, and were
immunogenic in rhesus monkeys. Monkeys immunized with a tetravalent
vaccine (ChimeriVax-DENV1–4) seroconverted to all four viruses after
one dose (except 1 of 6 did not seroconvert to DENV4). Preexisting immu-
nity from YF 17D vaccination (YF-VAX) did not significantly affect the
neutralizing antibody response.
A phase 1 trial found the safety profiles of YF-VAX and ChimeriVax-
DENV2 were similar, and no SEA were observed (Guirakhoo et al.,
2006). All recipients seroconverted to DENV2 after vaccination with 5
log10 PFU of the vaccine, and preexisting immunity to YFV did not inter-
fere with DENV2 seroconversion. In fact, all YFV-immune subjects also
seroconverted to the other DENV serotypes, whereas seroconversion to
the other serotypes was low in YFV-naive subjects.
352 Lauren E. Yauch and Sujan Shresta
Vaccine lot viruses of ChimeriVax-DENV1–4 were made using current
good manufacturing practice (cGMP) (Guirakhoo et al., 2004). Neu-
rovirulence was tested in cynomolgus monkeys after i.c. inoculation with
the tetravalent vaccine and was found to be reduced compared with
YF-VAX vaccination. Vaccine induced-protection was also tested in
cynomolgus monkeys. Monkeys received a single immunization s.c. with
a high or low dose (3 or 5 log10 PFU of each vaccine strain) of the tetravalent
vaccine and were challenged with wild-type DENV strains 6 months later.
All monkeys seroconverted to all four serotypes, and 22/24 were protected
from challenge.
Viral interference was studied in cynomolgus monkeys vaccinated with
the chimeric vaccine strains (Guy et al., 2009). Interference was observed in
monkeys given equivalent doses of each chimeric vaccine strain, with
DENV4 dominating, and several approaches were investigated to overcome
the interference. Immunization with bivalent vaccines at separate sites with
different draining lymph nodes, preexisting flavivirus immunity, decreasing
the dose of the dominant serotype, and boosting at 1 year all improved the
development of a balanced antibody response.
The ChimeriVax strains were highly attenuated for A. albopictus and
A. aegypti mosquitoes in terms of infection and dissemination (Higgs
et al., 2006; Johnson et al., 2004). Growth of the vaccine strains was also
studied in human myeloid DC and hepatic cell lines in vitro (Brandler
et al., 2005). The vaccine strains were not attenuated for replication in
DC compared with wild-type DENV or YF 17D but replicated to lower
titers than YF 17D in HepG2 and THLE-3 cells (but not HuH-7 cells),
suggesting the vaccine strains may be less hepatotropic than YF 17D and
therefore have less risk of inducing the hepatic failure that has been occasion-
ally been observed after YF 17D vaccination. Importantly, the chimeric
viruses were found to be genetically and phenotypically stable throughout
the manufacturing process (Mantel et al., 2011; Monath et al., 2005).
A tetravalent vaccine (TDV), containing�5 log10 tissue culture infective
doses (TCID50) of each recombinant serotype, was tested in flavivirus-naive
adults (Morrison et al., 2010). Two groups of 33 volunteers received the
vaccine at 0, 4, and 12–15 months or saline for first injection followed by
two doses of the TDV. The vaccine was safe, with no vaccine-related
SAE. Low-level viremia was observed primarily after the first dose and
was mainly DENV4. Each dose of the vaccine increased neutralizing anti-
body titers, and all volunteers receiving three doses seroconverted to all four
serotypes. The TDV was tested in children and adolescents (2–5, 6–11, or
353Dengue Vaccines
12–17 years of age) and adults in a nondengue endemic area (Mexico City)
(Poo et al., 2010). Subjects received three doses at 0, 3.5, and 12 months or
YF-VAX followed by two doses of TDV. The vaccine was safe, with no
vaccine-related SAE reported, and immunogenic. Seropositivity against
each serotype after three doses of TDV ranged from 77% to 92% and from
85% to 94% in the YF/TDV recipients. A phase 1 trial was then conducted
in the Philippines, a dengue-endemic country (Capeding et al., 2011). Chil-
dren, adolescents, and adults received three doses of the TDV vaccine at 0,
3.5, and 12 months. Reactogenicity was similar in adults and children, with
headache, injection site pain, fever, and myalgia most frequently reported.
A low level of viremia (primarily DENV4) was detected in some recipients,
most frequently after the first dose. After three doses, 100% of adults
seroconverted to all four serotypes, and seroconversion ranged from 83%
to 100% in children/adolescents. CD8þ T-cell responses against YF 17D
NS3 and DENV-specific CD4þ T-cell responses were detected in volun-
teers vaccinated with the tetravalent chimeric vaccine (Guy et al., 2008).
IFN-g dominated over TNF for both CD4þ and CD8þ T-cell responses.
After one vaccine dose, responses were serotype-specific and dominated by
DENV4 but broadened after a booster immunization.
A phase 2a study was designed to examine the safety and efficacy of TDV
vaccination in flavivirus-immune individuals (Qiao, Shaw, Forrat, Wartel-
Tram, & Lang, 2011). One dose of the TDV was given to persons who had
been vaccinated with monovalent live attenuated DENV1 or DENV2 vac-
cines, or YF-VAX 1 year prior, or flavivirus-naive adult volunteers. Prior fla-
vivirus immunity did not increase reactogenicity or the incidence of viremia,
but it did increase immunogenicity. In flavivirus-naive recipients, the neutral-
izing antibody response after one dose of TDVwas directed predominantly to
ients a more balanced neutralizing antibody response was observed.
A phase 2 study was conducted in 199 children (2–11 years of age) in
Peru who had varying levels of preexisting flavivirus immunity from YF
vaccination (Lanata et al., 2012). Children received 3 doses of TDV at 0,
6, and 12 months. The reactogenicity observed was similar to previous stud-
ies; injection site pain, headache, malaise, fever were most commonly
reported and decreased with subsequent vaccinations. No vaccine-related
SAE were reported. Viremia was detected in 44% of the 97 individuals
tested and was mainly DENV4. Vaccination was immunogenic as well
and resulted in 94% of recipients seroconverting to all four DENV serotypes
with comparable neutralizing antibody titers to the four serotypes.
354 Lauren E. Yauch and Sujan Shresta
Results of a phase 2b study of TDV were reported in 2012. The CYD-
TDV vaccine was given to children 4–11 years of age in dengue-endemic
Thailand (Sabchareon et al., 2012). The primary analysis included data
from 2452 vaccine recipients and 1221 controls. More than 90% of the
children had preexisting antibodies against DENV or JEV, and 70% were
seropositive against at least one DENV serotype. Three injections of the
vaccine were given at 0, 6, and 12 months, and the subjects were followed
for 13 months after the last dose. The vaccine was safe with no vaccine-
related SAE and immunogenic. Neutralizing antibody titers increased after
one dose and increased further after the second and third doses and
then decreased 1 year later. However, the overall protective efficacy in
preventing symptomatic dengue infection was only 30.2%. The efficacy
for the individual serotypes was 55.6% for DENV1, 9.2% for DENV2,
75.3% for DENV3, and 100% for DENV4. DENV2 was the most com-
mon infecting serotype, which skewed the overall efficacy. The antibody
neutralization data did not correlate with protection, as neutralizing anti-
body titers (measured by PRNT50) increased after each dose and were
highest against DENV2 and DENV3, yet the subjects were not protected
against DENV2 infection. The authors suggest in the future performing
neutralization studies on cells that express FcR, which are targets of DENV
in vivo. The PRNT also does not distinguish between balanced neutralizing
antibody responses to the four serotypes, or less protective cross-reactive
responses. In addition, antibodies have other functions besides neutraliza-
tion, including ADCC, which may be important for protection. Another
potential reason for the low efficacy includes an antigenic mismatch
between the DENV2 vaccine strain and the DENV2 strain that resulted
in infections. Finally, the lack of a DENV-specific T-cell response may
have contributed to the poor efficacy, as these chimeric vaccines consist
of YFV, not DENV, nonstructural proteins, which are the dominant tar-
gets of the anti-DENV T-cell response in humans and mouse models
(Weiskopf et al., 2013, 2011; Yauch et al., 2010).
Despite the disappointing protection observed, the study results were
informative andmay spur investigations that lead to the identification of cor-
relates of protection. Importantly, the vaccine was safe, with no vaccine-
related SAE induced, and there was no disease enhancement observed in
the presence of nonprotective immunity during the short duration of the
study. Phase 3 studies involving 30,000 individuals in Latin America and
Asia started in 2011 and will provide more data on the efficacy of this vaccine
(Clinicaltrials.gov NCT01374516 and NCT01373281).
355Dengue Vaccines
10. MOVING FORWARD
Years of dengue vaccine research have brought us close to the point of
having a licensed vaccine. Although the results of the CYD-TDV phase 2b
trial were disappointing, the findings were important in directing future vac-
cine development and will hopefully lead to the identification of immune
correlates of protection. The trial results highlighted the need to study
pre- and postvaccination immune responses in both flavivirus-naive and
flavivirus-immune individuals in more detail. The lack of efficacy against
DENV2 despite neutralizing antibodies measured by PRNT using Vero
cells suggests neutralization assays on cell types that express FcR may be
more relevant. In addition to examining neutralization, other antibody
functions can be studied as well. The titer, class, subclass, and avidity of anti-
bodies specific for E, prM, and NS1 can be determined. The ability of
vaccine-induced antibodies to mediate ADCC and fix complement can also
be analyzed. The magnitude, breadth, and functionality, including cytokine
production and cytotoxicity, of both CD4þ and CD8þ T-cell responses
should also be investigated. As mentioned earlier, recent studies point to
an important protective role for CD8þ T cells in the immune response
to DENV. Vaccines that induce robust T- and B-cell responses may prove
to be superior to those vaccines that induce robust antibody responses but
weak T-cell responses.
Overall, the vaccines currently in clinical trials are safe, and no disease
enhancement has been observed in vaccinated humans to date. However,
long-term studies, both in NHP and humans, are required to ensure waning
immunity does not predispose vaccinees to severe dengue disease. The
WHO recommends following subjects for approximately 3–5 years after
the last vaccination (WHO, 2011). Although no disease enhancement fol-
lowing DENV vaccination has been reported, recent studies of the human
antibody response to DENV found prM/M-specific antibodies are broadly
cross-reactive and weakly or nonneutralizing (Beltramello et al., 2010; de
Alwis et al., 2011; Dejnirattisai et al., 2010), suggesting it may be prudent
to minimize the anti-prM antibody response to avoid ADE.
Animal models provide the necessary tools for dissecting the mechanisms
of vaccine-mediated protection. As some of the vaccine studies discussed
earlier suggest that vaccine-induced immune responses differ in flavivirus-
naive versus flavivirus-immune individuals, animal models provide the tools
to evaluate vaccine-induced immune responses under well-defined naive
356 Lauren E. Yauch and Sujan Shresta
versus immune infection settings. Thus, vaccine-induced immune responses
in animal models of dengue disease should be studied in more detail, includ-
ing analyzing the magnitude and quality of the T-cell responses. The existing
murine and NHP animal models can also be improved, and/or new models
developed. Manipulating the virus or mouse immune system may lead to
more relevant models (Zompi & Harris, 2012). For instance, passaging of
DENV though monkeys may result in the isolation of a strain more virulent
for monkeys. Mice lacking only the type I IFN receptor may prove to be a
more relevant model than AG129 mice. In addition, adoptive transfer stud-
ies may be useful for studying subunit and inactivated vaccines. Wild-type
mice can be immunized with these nonreplicating vaccines, followed by
transfer of immune components from the vaccinated wild-type mice into
IFN receptor-deficient mice. The IFN receptor-deficient mouse models
serve as a stringent challenge assay, and the adoptive transfer system allows
for thorough analysis of vaccine-induced humoral versus cellular response in
normal mice.
The lack of an adequate animal model for evaluating live attenuated den-
gue vaccine-induced immune responses has prompted the development of a
dengue human challenge model (DHCM). In a recent study, subjects pre-
viously vaccinated with the WRAIR/GSK live attenuated tetravalent vac-
cine (TDV) were challenged with underattenuated DENV strains to
evaluate the safety of challenge with the underattenuated strains and to eval-
uate the relationship between vaccine-induced neutralizing antibody titers
and protection (Sun et al., 2013). Subjects who had received the TDV
12–42 months previously, or naive controls, were challenged with under-
attenuated DENV1 or DENV3. All 5 vaccinated subjects challenged with
DENV1 were protected, and 2 of 5 challenged with DENV3 were protec-
ted. The 4 naive control recipients developed DF upon challenge. Neutral-
izing antibody titers correlated with protection in all but 1 subject who was
protected from DENV1 challenge despite no detectable neutralizing anti-
bodies. The DENV3 challenge was associated with significant elevations
in AST/ALT. This study demonstrated the feasibility of human challenge
to evaluate DENV vaccine candidates. A DHCM workshop, sponsored
by the WRAIR and the NIH, was held in 2011, and the consensus was that
a DHCM could be developed safely, if appropriate challenge strains can be
identified and produced under cGMP (Durbin & Whitehead, 2013). Safety
is a major concern for a DHCM, as challenge of vaccine recipients with
underattenuated strains could put the subjects at risk for developing severe
disease. Additionally, there is no approved therapeutic that could be used to
357Dengue Vaccines
treat recipients who develop DF or DHF/DSS. However, a DHCM could
provide valuable information on the immune response to DENV and poten-
tially lead to the identification of immune correlates of protection.
A DHCM could also be useful for selecting vaccine candidates for field
studies.
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