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Development of sanofi pasteur tetravalent denguevaccineBruno
Guy, Melanie Saville & Jean LangPublished online: 01 Sep
2010.
To cite this article: Bruno Guy, Melanie Saville & Jean Lang
(2010) Development of sanofi pasteur tetravalent dengue
vaccine,Human Vaccines, 6:9, 696-705, DOI: 10.4161/hv.6.9.12739
To link to this article:
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review
Human vaccines 6:9, 696-705; September 2010; 2010 Landes
Bioscience
696 Human vaccines volume 6 issue 9
*Correspondence to: Bruno Guy; Email:
[email protected]: 05/28/10; Accepted:
06/16/10Previously published
online:www.landesbioscience.com/journals/vaccines/article/12739DOI:
10.4161.hv.6.9.12739
The Sanofi Pasteur tetravalent dengue vaccine candidate is
composed of four recombinant live attenuated vaccines based on a
yellow fever vaccine 17D (YFv 17D) backbone, each expressing the
prM and envelope genes of one of the four dengue virus serotypes.
Pre-clinical studies have demonstrated that the Tv dengue vaccine
is genetically and phenotypically stable, non-hepatotropic, less
neurovirulent than YFv 17D and does not infect mosquitoes by the
oral route. in vitro and in vivo preclinical studies also showed
that the Tv dengue vaccine induced controlled stimulation in human
dendritic cells and significant immune responses in monkeys. Tv
dengue vaccine reactogenicity, viramia induction and antibody
responses were investigated in three Phase i trials in the USA, the
Philippines and Mexico, in a two or three-dose regimen over a 12
month period. results showed that the majority of adverse events
were mild to moderate and transient in nature. viremia was
transient and low, and was not increased after initial dengue Tv
administration, even in the case of incomplete responses.
fSeropositivity (10 in a PrNT 50 assay) was 100% for all four
serotypes in flavivirus-naive adults injected with 3 doses of Tv
dengue vaccine in the USA. Similarly, seropositivity was 88100%
following three administrations in flavivirus-naive Mexican
children aged 25 years. Furthermore, the proportion of seropositive
subjects increased with each dengue Tv injection in the Philippines
where baseline flavivirus immunity was high. responses were also
monitored at the cellular level in humans, and their level and
nature were in good agreement with the observed safety and the
immunogenicity of the vaccine. Finally, the challenges inherent to
the development of such Tv dengue vaccines will also be discussed
in the last part of this review.
in conclusion, preclinical and clinical results support the
favorable immunogenicity and short-term safety of the den-gue Tv
vaccine. An extensive clinical development program for dengue Tv is
underway including completion of the enroll-ment of 4,000 411 years
old children in an efficacy trial in Thai-land, in an area of high
dengue incidence. Assuming continued successful outcomes, initial
submissions to regulatory authori-ties are envisaged within a
5-year period.
Development of Sanofi Pasteur tetravalent dengue vaccine
Bruno Guy,* Melanie Saville and Jean Lang
research and Discovery Departments; sanofi Pasteur; Marcy
letoile, France
Key words: dengue, vaccine, human, development, immunity
Introduction
Several members of the Flavivirus genus are serious threats to
human and animal health. Among them, dengue viruses represent a
major and growing medical problem. All of the four serotypes of
dengue virus can cause clinical manifestations ranging from
self-limiting dengue fever to severe dengue hemorrhagic fever (DHF)
and fatal dengue shock syndrome (DSS). The number of dengue
infections in endemic areas has continued to increase over the past
two decades. Over one hundred countries are affected with over
three billion people at risk (reviewed in ref. 1). It has been
esti-mated that more than 100 million dengue infections resulting
in 24,000 deaths occur annually. Children are the most affected.
This increased disease incidence and extended geographical reach of
dengue2 have made the development of an effective vaccine an
international health priority.
Academic laboratories and pharmaceutical companies have
developed several dengue vaccine candidates using a variety of
tech-nologies, including live-attenuated vaccines (LAVs),
recombinant virus vectors expressing dengue envelope (E) antigens,
recombi-nant proteins and DNA vaccines, none of which have been
licensed (for reviewes see refs. 35). Sanofi Pasteur is now
developing a tet-ravalent live attenuated chimeric dengue virus
vaccine based on the yellow fever 17D vaccine strain. The
technology (sometimes referred to as ChimeriVax) behind the
production of the chimeric dengue vaccine viruses originated at St.
Louis University,6 and was applied by Guirakhoo et al. at Acambis
Inc., (now a part of Sanofi Pasteur).7 As we will develop in this
review, this dengue vaccine candidate is immunogenic and safe in
humans, and is currently being evaluated in large scale efficacy
studies.
The live attenuated and chimeric nature of these vaccine viruses
necessitates extensive preclinical and clinical charac-terization,
from the early stages of research through to clinical development.
Furthermore, their status as genetically modified organisms (GMO)
required compliance with additional specific regulations. These
issues were reviewed in reference 8, focusing on the various tools,
assays and research strategies implemented as part of our chimeric
flavivirus research and development pro-gram, and will be discussed
only briefly here.
Construction of the Tetravalent Dengue Vaccine
The four monovalent chimeric vaccine viruses, CYD1-4, were
constructed by replacing the genes for YF vaccine (YFV 17D 204)
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ProDUcT review review
In vitro genetic stability. The YFV 17D vaccine genome is
remarkably stable, both in vivo and in vitro,10 which may be
attributed to the low error-rate of the virus RNA polymerase.11 The
same enzyme assures the viral replication of the CYD viruses and it
was thus expected that these viruses would dis-play a similarly
high stability. The full genome sequence of the each CYD was
established at various stages during the manufac-ture of GMP
vaccine lots, from the first passages, to premaster seed lots
(PMSL), master seed lots (MSL) and bulk and ulti-mately at a later
step in the process (bulk +10 passages). Each of the four CYD
viruses exhibited high genomic stability in cell culture, with a
total of only nine mutations observed. Five of these mutations were
detected only at late passages (between p10 and p21), and three of
them found in a mixed population with the original sequence. One
mutation was silent. All muta-tions except one in the E gene were
located in the non-struc-tural regions of the genome and likely
reflect adaptation to Vero cells. The non-silent mutations present
at early manufactur-ing steps appeared during the scale change
between PMSL to MSL and were then conserved stably throughout the
process (unpublished data).
In vitro phenotypic stability. Consensus genome sequencing is
unsuitable for the detection of minor, quasi-species in a vac-cine
seed or batch and gives little information about the poten-tial
biological consequences of a particular mutation. However,
premembrane (prM) and envelope (E) proteins, with those of the
four dengue serotypes6,7,9 (Fig. 1). A tetravalent CYD1-4 dengue
vaccine (TDV) is produced by combining the four monovalent viruses
in a single vaccine preparation.
Preclinical Evaluation
Before entering clinical trials, all vaccine candidates must be
tested for safety and immunogenicity in preclinical studies. For
these dengue vaccines, testing can be conducted both in vitro on
primary or transformed cells, including human cells, and in vivo in
animals, in particular non-human primates (NHP). Preclinical
studies were designed to provide information on the phenotypic and
genotypic stability of the vaccine candidates, their tropism,
structure, ability to replicate and to be transmitted by mosquito
vectors, as well as to document specific aspects linked to the use
of genetically modified organisms (GMOs). All of the above directly
or indirectly affect safety and immunogenicity.
It is also important that a dengue vaccine provides protective
immunity against all four circulating viral serotypes, a point that
was also addressed in preclinical studies. Figure 2 presents the
different types of cells and tissues potentially affected by
den-gue vaccines and Figure 3 shows some types of preclinical
stud-ies that can be carried out to study the consequences of such
interactions.
Figure 1. construction of the four chimeric vaccines.
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their good clinical safety profile and immunogenicity (see
below).These results were further confirmed and extended using DNA
array profiling (Guy B et al. J Infect Dis; In Press).
The tropism of flaviviruses is mostly linked to the envelope, so
CYD should not exhibit the same tropism as YFV 17D. To test this
hypothesis, the growth kinetics of CYD1-4 and paren-tal viruses
(wild-type DEN-1-4 and YF 17D) were assessed in three hepatic cell
linesHepG2, Huh7 and THLE-3as a potential marker of viscerotropism.
Replication of all chime-ric viruses in HepG2 and THLE 3 cells, but
not in Huh7, was markedly lower than that of YF-VAX.16 Differences
in findings between cell lines may be explained by the fact that
Huh7 cells are permissive to replication of many viruses,
irrespective of their attenuated phenotype. These results
nevertheless suggest that the CYD1-4 viruses are less hepatotropic
that YF 17D virus vaccine in humans.
Other assays which will not be described here have also been
used to further characterize the CYD vaccine candidates. These
include: electron microscopy to assess viral maturity; SDS/PAGE
analysis to assess the consistency of the protein content and
pro-file of the vaccines, glycosylation status; replication in DC
SIGN-transfected cell lines to assess the ability of vaccine
candidates to interact with this molecule, and subsequently
effectively enter cells; replication in insect C6/36 cells and
temperature sensitiv-ity assays.
mutations that affect the infection efficiency, growth,
penetration or spread of the virus in cell culture, generally
modify the plaque phenotype.12,13 Phenotype consistency was
therefore monitored throughout vaccine lot production using a
plaque size phenotype assay. By measuring at least 100 plaques, it
was estimated that we achieved a greater than 90% probability of
detecting revertants representing 2% of the total population. We
found the plaque size phenotype of all four CYD viruses to be
stable at all pro-duction steps. Phenotypic stability was also
assessed in animal models when available (see below).
In vitro preclinical studies. Skin dendritic cells (DCs) are
among the first cells to encounter virus after inoculation and are
also the most efficient antigen-presenting cells (APC) impli-cated
in the primary immune response. Interactions between human DCs and
wild-type (wt) dengue viruses have been well documented,14,15 it
was therefore interesting to compare immune consequences of
infection with attenuated vaccine viruses ver-sus their wt parents.
We investigated the infectivity of the four CYD viruses in
monocyte-derived human DCs,16 as well as the consequences of
infection in terms cellular activation and mat-uration and the
secretion of pro- and anti-inflammatory cyto-kines, chemokines and
type I interferons.17 The CYD1-4 viruses were seen to induce DC
maturation and a controlled response, accompanied by limited
inflammatory cytokine production and consistent expression of
anti-viral type I IFN, in agreement with
Figure 2. innate and adaptive immune responses potentially
triggered by dengue vaccine candidates. Upon vaccination, dengue
vaccine viruses may replicate preferentially in cells from the
monocytic lineage, such as monocytes and dendritic cells (Dcs).
Antigen presentation to cD4/cD8 T cells will trigger their
activation and subsequent B-cell activation. Activated memory B
cells can also constitute potent antigen-presenting cells upon
boost-ing. it is also of interest to monitor the potential
replication and consequences, of dengue vaccines in other cell
types, including endothelial cells and hepatic cells.
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neutralizing antibodies.9,20,21 In particular they have been
used to explore immunization parameters such dosing regimen and the
vaccine formulation, and to monitor for viral interference between
the four vaccine viruses. As absolute thresholds for pro-tective
immunogenicity and acceptable viremia are difficult to establish,
and cannot necessarily be extrapolated between host species, immune
responses and viremia induced by vaccine can-didate viruses are
compared with those induced by wt viruses. NHP studies have shown
that primary immunization with TDV induced a short-lived, low-level
viremia, which was not present after booster immunizations. One or
several injections of TDV conferred immunity against the four wt
dengue serotypes, and almost complete protection against each
serotype upon subse-quent wt challenge.9
As with any multivalent vaccine, dengue vaccine development is
complicated by the potential for interference between sero-types
which can result in a dominant immune response against only one or
two serotypes. In NHPs, interference was observed after vaccination
with a tetravalent preparation containing 5 log
10
CCID50
of each virus (designated 5,555, and considered as the reference
tetravalent vaccine preparation).22 We identified several
approaches to mitigating interference: (1) simultaneous
adminis-tration of two complementary bivalent vaccines at separate
ana-tomical sites drained by different lymph nodes; (2) sequential
administration of two complementary bivalent vaccines; (3)
pre-immunization with a heterologous flavivirus; (4) adaptation of
formulations by decreasing the dosage of the immunodominant
In vivo preclinical studies. Mouse models of neurovirulence have
been used to discriminate between the neurotropism of den-gue
vaccine candidates with that of their parental viruses. One such
model in suckling mice has been shown to be an acceptable
alternative to NHP models.18 After i.c. inoculation in both mice
and NHPs, all four CYD viruses were significantly attenuated, even
compared with the parental YFV 17D vaccine. The suck-ling mouse
model is now routinely used for in-process control testing during
the manufacturing process of Sanofi Pasteurs YFV 17D-based
flavivirus vaccines.
Some NHPs, including rhesus (Macaca mulatta) and cyno-molgus
monkeys (Macaca fascicularis), are sensitive to dengue and YF
infections. The World Health Organization (WHO) rec-ognizes these
species as good models for the assessment of the neurotropism and
the viscerotropism of attenuated YF vaccines (WHO Technical report
series, N 872, 1998). Viremia can be used to assess the attenuation
of vaccine candidates by comparing vaccine virus viremia with that
of the wt parental strains. As den-gue-infected monkeys do not
develop disease symptoms, viremia can also be used to assess
protection against wt viral challenge by evaluating the reduction
in wt virus viremia in vaccinated animals compared with
unvaccinated control animals. Viremia can thus be considered as
both a direct indicator of tropism and an indirect indicator of
safety since it has been identified as one of the factors
associated with virulence and disease severity in humans.19
NHP studies can also be used to provide additional infor-mation
on the ability of dengue vaccine candidates to elicit
Figure 3. Preclinical assays used to characterize dengue vaccine
candidates.
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700 Human vaccines volume 6 issue 9
To assess whether mosquitoes could become infected with the CYD
viruses from feeding on a vaccinated host, the ability of CYDs to
replicate in Aedes albopictus mosquito cell culture (C6/36) and in
Ae. aegypti mosquitoes, the principal vectors of YF and dengue
viruses, was evaluated in comparison with the parental YF 17D and
wt dengue viruses.23 The CYD viruses were unable to infect orally
Ae. aegypti and Ae. Albopictus or to repli-cate in midgut tissue
after intra-thoracic inoculation, and were more attenuated than YF
17D virus in these species. Together with the absent or low-level,
short-lived CYD viremia in human vaccinees, the inability to infect
and replicate in mosquitoes vec-tors are critical safeguards
against the dissemination of CYD viruses in the environment.
Based on theoretical assumptions and an analogy with
non-flavivirus vaccines, it was suggested that new viruses might
emerge from the recombination between flaviviruses or with
dissimilar RNA viruses,24 although the authors assumptions were
challenged by most experts in the field.25,26 A recent study
investigated the likelihood of intermolecular recombination between
different flaviviruses using pairs of replicons derived
serotype 4 CYD virus and (5) administration of a booster at 1
year. These studies also showed that immunizations should be spaced
several months apart to prevent negative interference, possibly due
to short-lived cross-reactive (IgM) antibodies, cross-reactive T
cells or to innate immunity, and to allow a better induction of
memory. Such regimens have been tested in humans in different
trials and have confirmed the importance of a one year booster.
These studies have also highlighted the differences between
species, as the interval between sequential immuniza-tion with
complementary bivalent vaccines needs to be longer in humans than
in monkeys (unpublished data).
Environmental Risk Assessment
A number of theoretical issues associated with the live and GMO
nature of the CYD viruses have been addressed throughout their
development as reviewed in detail in reference 8. The four most
frequently raised concerns are: transmission by arthropod vec-tors,
recombination with a circulating virus, reversion to viru-lence and
risks of viscerotropism.
Figure 4. GMT (log10 with 95% ci, wHo reference strains) in nave
volunteers obtained after 1 to 3 immunizations of Tv dengue vaccine
(from ref. 32). Group 1: 3 doses of TDv given at D0, M4 and M12.
Group 2: one dose of placebo given at D0 followed by 2 doses of TDv
given at M4 and M12. Blood samples were taken prior to each dose of
vaccine and 28 dose post vaccination.
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varies between regions, as do the current recommended
vacci-nation schedules, meaning trials will have to be conducted in
parallel on different continents potentially with different
co-administered vaccines.
In addition to compliance with Good Clinical Practice
guide-lines and all applicable national and international
regulations, clinical trials are being designed and conducted in
accordance with the WHO Guidelines for the Clinical Evaluation of
Dengue Vaccines in Endemic Areas (WHO/IVB/08.12). Indeed, our first
priority is to develop the vaccine in endemic countries of
Asia-Pacific, Latin America and the Caribbean to address the unmet
medical need for children and adults. Trials will also be performed
in non-endemic countries, for example in Europe and USA, for
travelers and military personnel.
Phase I clinical evaluation. The first clinical evaluation of a
CYD vaccine candidate was with a monovalent serotype 2 CYD virus
(CYD-2). The safety profile of CYD-2 was reported to be similar to
the YF-17D control (YF-VAXTM) and transient low-level viremia was
observed. Most participants seroconverted to dengue virus strain
16,681, and prior YFV 17D immunity was found to induced stronger,
broader (cross-protective) and long lasting anti-dengue immune
responses.31 As of April 2010 more than 4,800 volunteers have
received at least one dose TDV (5 log
10 CCID
50 of each serotype), including children from 2
years of age to adults up to 45 years, in both dengue-endemic
(Philippines, Thailand) and non-endemic (USA, Mexico City,
Australia) areas.
The first study showing complete seropositivity (10 in a PRNT 50
assay) against the 4 dengue serotypes was carried out in
dengue-nave US adults who received 3 doses in a 03.512 month
regimen or 2 doses 89 months apart.32 Consistent with the TDVs
favorable safety profile (Table 1), viremia was low and was mainly
CYD-4 after the first vaccination. After the second TDV
vaccination, almost no viremia was detected by either RT-PCR or
plaque assay (PA) for any serotype. After the second vaccination,
more than 85% of vaccinees had no detect-able CYD-1, 2 or 3
viremia. This finding has significant impli-cations for the safety
of this vaccine as is shows that while the first TDV vaccination
did not elicit complete seroconversion to serotypes 1, 2 and 3,
this did not induce sensitization to a second TDV vaccination in
the presence of heterologous anti-serotype 4 antibodies. All
participants who received three TDV vaccinations seroconverted to
all four WHO reference strains tested (Fig. 4). An increased immune
response with an increased number of doses was apparent: both GMT
and the percentage of seroconverted participants increased after
each vaccination. The first vaccination induced a neutralizing
humoral response mainly against serotypes 4 and 2, and to a lesser
extent against 1 and 3. The second and third doses increased the
percentage of seroconverted participants as well as the GMTs for
all four serotypes, balancing the immune response across all four
sero-types (GMT of 67, 538, 122 and 154 against serotypes 1, 2, 3
and 4 respectively). Moreover, there was a trend towards higher
GMTs and higher seroconversion rates after 2 TVD doses in
vol-unteers receiveing these doses 89 months rather than 3 months
apart (especially for serotypes 1 and 3). This latter
observation
from Tick-borne encephalitis virus (TBVE), Japanese
encepha-litis virus (JEV) and West Nile virus (WNV).27 The very few
recombination events detected (none for TBEV or WNV), were aberrant
recombinations resulting in virus with impaired growth properties.
Results showed that flaviviruses have a low propen-sity for
homologous recombination. The findings complement those of previous
studies investigating what would happen in the unlikely event that
such a recombinant virus emerged. We considered the worst-case
scenario where one of the attenuated CYD vaccine viruses recombined
with the wt YF Asibi virus. All observations of replication and
transmission in mosquitoes, as well as outcomes in NHPs showed that
these recombinants were attenuated compared with the parental wt
viruses.28,29 These studies also suggested that the chimerization
process itself con-tributed to the attenuation of these viruses.
Thus, not only is the recombination of the CYD vaccine viruses with
a wt flavivirus extremely unlikely, any recombinant would be
unlikely to cause disease or be disseminated.
While reversion to virulence has been raised by some as a
potential concern, reversion of a CYD virus into a virulent YF
virus is virtually impossible given the genetic stability discussed
above. In addition to not having YFV 17D preM or E genes, the CYD
chimeric virus features numerous attenuating residues within the
seven YFV 17D nonstructural genes and the capsid protein gene.
Reversions in all of these would be required for virulent virus to
emerge.
Although extremely rare, acute viscerotropism disease has
occurred following YF 17D vaccination (estimated incidence: 0.30.4
per 100,000 vaccinated individuals).30 There is a per-ceived risk
that such a serious adverse event might therefore occur after CYD
vaccination. However, as stated previously, the viral tropism and
virulence are largely linked to the E protein, and the E gene is
precisely one of the YFV 17D genes that is not present in the
chimeric dengue viruses. Additionally, as described above, in vitro
and preclinical in vivo experiments have shown that the
viscerotropism and neurotropism of CYD viruses are signifi-cantly
attenuated compared with YFV 17D. It is thus plausible to propose
that the safety profile of chimeric dengue vaccines will be
improved over that of YFV 17D, particularly with respect to most
neurotropic adverse events.
Clinical Development
The considerations and challenges of clinical development
include: (1) the need to induce an adequate and balanced immune
response to all four serotypes; (2) the need for two or three
vac-cinations over a period of up to 12 months in flavivirus-nave
individuals; (3) the current absence of a correlate and threshold
of protection and thus the need to demonstrate clinical efficacy;
(4) the need to demonstrate long term safety and immunoge-nicity;
(5) the theoretical risks of sensitization to severe dengue
infection (DHF) after vaccination and of acute viscerotropic
dis-ease (AVD) and neurotropic disease (AND) that are very rare
serious adverse events after YFV 17D vaccination and (6) the need
to comply with GMO regulations. Additional complexity is brought by
the fact that the flavivirus immunological background
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geographically diverse strains, and similar analyses are ongoing
with human sera.
So far the CYD vaccine candidates have demonstrated a
satis-factory safety profile. No serious adverse events (SAE)
related to vaccination have been identified in the studies
mentioned above. Unlike some whole virion live-attenuated dengue
vaccine can-didates, the CYD vaccine does not appear to induce the
mild dengue-like syndrome associated with viremia.4 Biological
safety results have been unremarkable and reactogenicity has
appeared similar to that of the control vaccines. Reactogenicity
was not increased by the presence of baseline immunity to either
dengue or yellow fever, nor was it increased after the second or
third vac-cination than after the first.
While protection against dengue is associated with both humoral
and cellular responses, these responses have also been implicated
in the immunopathology of severe dengue disease (reviewed in ref.
37). On the antibody side, it was hypothesized that one of the
mechanisms responsible for severe disease is the enhancement of
viral replication by heterotypic, non-neutral-izing antibodies from
a prior infection (via the Fc receptor on mononuclear leukocytesthe
antibody dependent enhance-ment (ADE) phenomenon38,39). This point
will be specifically addressed below. On the cellular side, immune
responses should include high-avidity homologous multivalent
responses against all serotypes, should be Th1-biased and dominated
by IFN over TNF. In particular, it has been shown that
heterologous, cross-reactive responses tend to trigger TNF while
homologous responses trigger IFN37,40,41
CD4 and CD8 immune response against the parental YF 17D and
dengue viruses elicited by CYD1-4 vaccination were thus assessed in
volunteers with and without pre-existing flavivirus immunity.42 The
TDV triggers no detectable changes in serum pro-inflammatory
cytokines, regardless of the baseline immune status, but induced
significant YFV-17D NS3-specific CD8 responses and dengue virus
serotype-specific T helper 1 responses, dominated by IFN over TNF.
As for antibodies, responses were initially dominated by serotype 4
in baseline-nave individ-uals, but subsequent vaccinations
broadened the serotype-specific
confirmed observations in monkeys immunized with the same TDV,
where marked increases in immune responses against all serotypes
were seen when there was a 10 month interval between the second and
third vaccinations, but not when this interval was only two
months.22 Cellular immune responses were also moni-tored in this
clinical trial. The observed level and nature (cyto-kine profile,
CD8/Th bias, serotype dominance) of innate and adaptive cellular
responses were in good agreement with both the favorable safety
profile and humoral immunogenicity data (see below).
Follow-up studies with the same vaccination regimen as described
above were performed in children as young as 2 years as well as
older children, adolescents and young adults (up to 45 years) in
both non-endemic (Mexico City) and dengue endemic areas
(Philippines).33,34 Findings from these studies are consistent with
those in the study described above conducted among US adults32 and
further show that: flavivirus preimmunity (against Yellow-fever 17D
vaccine in Mexico or Japanese encephalitis and dengue in the
Philippines) has a positive impact on den-gue vaccine
immunogenicity without any negative effects on safety; three TDV
vaccinations with a 03.512 month sched-ule induced robust antibody
responses against all four serotypes; and that levels of vaccine
virus viremia are consistently low (usu-ally lower than lower limit
of quantitation of the PCR or plaque assays used) in all
populations.
In another third study in Australian adults prior immunity
against either dengue 1 or 2 serotypes (conferred by vaccination
with conventionally attenuated dengue vaccine candidates devel-oped
by Mahidol University, Bangkok, Thailand) primed for a strong and
broad response to TDV vaccination against all four serotypes.35
It was also important to address the potential risk that a
circu-lating virus escapes vaccine-induced immunity. The capacity
for sera raised against the CYD vaccine viruses to cross-neutralize
a large panel of wt strains from each of the four dengue serotypes,
collected recently from different areas of dengue endemicity has
also been investigated, first with monkey sera.36 Results obtained
point to broad coverage of vaccine-induced antibodies against
Table 1. Adverse event (Ae) observed after TDv administration in
nave volunteers (from ref. 32)
V1 D0
V2 M4
V3 M12
Group 1 1st TDV (N = 33)
Group 2 Placebo (N = 33)
Group 1 2nd TDV (N = 30)
Group 2 1st TDV (N = 33)
Group 1 3rd TDV (N = 23)
Group 2 2nd TDV (N = 26)
n (%) n (%) n (%) n (%) n (%) n (%)
Any Adverse event 28 (84.8) 22 (66.7) 24 (80.0) 24 (72.7) 15
(65.2) 17 (65.4)
Any Adverse reaction 27 (81.8) 20 (60.6) 21 (70.0) 21 (63.6) 10
(43.5) 17 (65.4)
Solicited injection site reaction 6 (18.2) 5 (15.2) 11 (36.7) 8
(24.2) 4 (17.4) 7 (26.9)
Solicited Systemic reaction 26 (78.8) 19 (57.6) 20 (66.7) 18
(54.5) 10 (43.5) 16 (61.5)
Severe adverse event 6 (18.2) 2 (6.1) 1 (3.3) 4 (12.1) 0 (0.0) 0
(0.0)
Severe solicited reaction 5 (15.2) 2 (6.1) 1 (3.3) 4 (12.1) 0
(0.0) 0 (0.0)
injection site 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0
(0.0)
Systemic 5 (15.2) 2 (6.1) 1 (3.3) 4 (12.1) 0 (0.0) 0 (0.0)
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able to induce a neutralizing response against all four
serotypes should circumvent the issue.
As stated by WHO guidelines, consensus exists that clinical
development of dengue vaccines should not be forestalled by
cer-tain hypothetical safety concerns.45 The next round of large
effi-cacy trials with a sample size of several thousand (Phase 3)
will provide further evidence of any safety issues occurring in
vaccin-ees versus controls. However, it is likely that this
theoretical risk will only be evaluable by Phase 4 trials and
post-marketing sur-veillance, due to the very rare occurrence of
such severe outcome.
Future Challenges
In addition to the direct challenges of clinical development,
and as the sanofi pasteur TDV enters clinical phase III, it is
important to set appropriate goals. Different additional challenges
need to be met to ensure the vaccines successful licensure and
implemen-tation in the field.
Immunology and non-clinical research challenges. We need to
better understand the protective mechanisms induced by den-gue
vaccine candidates. Future clinical efficacy trial data can be used
to retrospectively benchmark the preclinical models dis-cussed
above, as well as potentially to derive a correlate of protec-tion
which would be of great interest. Bridging between NHP data and
clinical protection, might facilitate the future develop-ment of
new vaccine formulations or technologies by reducing the need for
further expensive and lengthy clinical trials. With this in mind, a
clear immunologically-driven strategy to develop robust,
high-throughput, standardized assays is warranted to aid clinical
development.
Firstly there is a need for high-throughput serotype specific
neutralization assays as well as standardized, reproducible
cell-mediated immunity assays that can be used with small volumes
blood, and thus be suitable for application in pediatric studies
(see above). A large array of questions related to the evaluation
of cellular responses was discussed during a WHO task force on
dengue cellular immunity in Bangkok in 2007, generating gen-eral
recommendations.46 It was stated in particular that, although not
mandatory for registration, documenting such responses in clinical
trials, including phase 3 trials, would be recommended and would
bring important information relative to the short-term and
long-term safety and immunogenicity of the vaccine candidates.
Secondly, reliable and simple assays to examine antibody-binding
affinity and kinetics might provide useful estimates of the overall
avidity of sera against viral or E protein preparations at the
polyclonal level. This approach has already been investi-gated by
some investigators, and it would be of interest to com-pare overall
avidity of responses induced by vaccination versus that induced by
infection, as well as to compare overall avidity with
neutralization antibody results. Isotyping of E-specific IgG
subclasses might also be of interest, as factors such as their
com-plement fixing activity could be of importance.
Thirdly understanding interference mechanisms between dengue
viruses, might lead to simpler vaccination regimens com-pared with
the currently considered regimen, involving multiple
responses. A similarly broad response was seen after primary TDV
vaccination in participants with preexisting dengue sero-type 1 or
2 immunity. Data also demonstrated an absence of cross-reactivity
between YF 17D and dengue NS3-specific CD8 responses, and enabled
us to identify three new CD8 epitopes in the YF 17D NS3 antigen.
According to the original antigenic sin hypotheses, suboptimal,
heterologous, anti-NS3 CD8 responses may be involved in the
severity of secondary heterologous infec-tion.43 We observed an
almost complete absence of CD8 cross-reactivity between YF 17D and
dengue NS3 antigens, meaning that no potentially deleterious
cross-serotype anti-NS responses would be induced by YF17D
NS3-expressing CYDs. Subsequent natural infection would therefore
boost dengue-specific immune responses with a non-deleterious
profile.37,40,41
From a practical point of view, one can mention that the
anal-yses in these studies required a significant amount of blood
(from 35 to 50 ml) and would not be applicable to infants or
children. We are currently exploring the possibility of performing
analyses on a limited (up to 3 mL) amount of blood, in order to be
able to analyze responses in children.
Phase II and III clinical evaluation. Clinical phase II trials
are being conducted on several continents to assessing the safety
and immunogenicity of the TDV in children, adolescents and adults
with a diverse flavivirus infection and vaccination history and to
investigate co-administration with another live virus
vac-cinemeasles, mumps, rubella vaccinein toddlers. A proof of
concept efficacy trial is also underway as part of phase IIb. Four
thousand Thai children aged 411 years will receive 3 sub-cutaneous
injections of either TDV or control vaccine at 0612 months and will
be followed to assess efficacy against virologi-cally-confirmed
dengue disease, regardless of the severity. This will be followed
by large phase III trials in children and adoles-cents in Asia and
Latin America.
In compliance with the WHO guidelines, at each phase of clinical
development many subjects will be followed up long term (35 years).
This will allow us to monitor long term safety including potential
severe Dengue, and to assess antibody persis-tence and the
potential need for a booster dose.
Antibody Dependent Enhancement
The etiology of DHF appears to be multi-factorial. Whether or
not antibody dependent enhancement (ADE, see above) is one of these
factors in vivo is still a matter of debate (reviewed in ref. 8).
We nevertheless took these potential concerns into account and
developed early in our DENV vaccine research a sensitive and
reproducible in vitro assay using FcRII positive-K562 cells and
flow cytometry. Sera from Thai children vaccinated with
first-generation LAV candidates were analyzed using this assay and
correlated with a low/absent risk linked to ADE activity in vitro,
despite very diverse immune profiles, from low to high PRNT levels
against one or several dengue serotypes.44 Specifically, in vitro
ADE was absent in the presence of broad neutralizing response
against all four DENV serotypes. Thus, whatever the role of ADE in
the etiology of severe dengue in vivo, a vaccine
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704 Human vaccines volume 6 issue 9
data, identify new directions and establish useful synergies and
networks to allow dengue scientists and vaccinologists to make
significant advances. Efforts such as this need to be renewed, in
order to help establish regional scientific networks for
den-gue-endemic countries. Furthermore, stronger global alliances
between the WHO, PAHO, national regulatory authorities, key opinion
leaders, leading scientists, private foundations and vac-cine
developers are needed to share data, regulatory, risk man-agement
and licensure requirements in a new partnership model rather the
usual private versus public paradigm.
Conclusions
The Sanofi Pasteur tetravalent live attenuated chimeric virus
den-gue vaccine candidate has demonstrated satisfactory safety and
immunogenicity in both in vitro and in vivo pre-clinical tests, as
well as in clinical trials in both flavivirus-nave and immune
individuals. Potential risks, however unlikely, hypothesized as
being associated with these chimeric viruses have been explored in
depth. Both humoral and cellular responses are induced in humans
against all four serotypes and long-term follow-up will address the
duration of immunity and theoretical long-term safety issues. An
effective vaccine is now urgently required against dengue, and with
the initiation of large scale efficacy tri-als, the present vaccine
candidate provides hope that protection is now within our
reach.
Acknowledgements
The authors would like to acknowledge Grenville Marsh for
editorial assistance, all sanofi pasteur and former Acambis team
members, as well as external collaborators involved in
preclini-cal, clinical and industrial development, in particular
Farshad Guirakhoo, Tom Monath, Steve Higgs, Sutee Yoksan, Veronique
Barban, Anke Harenberg, Remi Forrat, Denis Crevat, Gustavo Dayan,
Anh Wartel-Tram, Betzana Zambrano, Enrique Rivas and Rafaele Dumas.
We would also like to acknowledge all investigators, in particular
Drs. Dennis Morrison, Maria Rosario Capeding, Jorge Luis Poo, J
Qiao and D Shaw, as well as the volunteers involved in the clinical
trials.
doses tetravalent vaccine given over 6 to 12 months. Applied
preclinical research is therefore needed to (1) better understand
the mechanisms of interference involving innate and/or adaptive
immunity in suitable in vitro and in vivo models (e.g., through the
assay developments mentioned above), (2) use this knowl-edge to
develop new regimens requiring fewer doses over shorter periods and
(3=) develop new vaccine technology strategies to alleviate
interferences without compromising immunogenicity and safety.
Fourthly, non-clinical safety models need to be further
explored. While satisfactory mouse and NHP models exist for
neurotropism, a similar model for viscerotropism is lacking. Such
models could either mimic the immune profile elicited by wt dengue
viruses and allow benchmarking of (non-human) safety,
biodistribution and protective activity of dengue vaccine
candi-dates or alternatively serve as true pathogenic models for
dengue disease, although this latter goal may not be
achievable.
Institutional and resource challenges. Until recently, inter-est
and investment in dengue disease and vaccination was sig-nificantly
less than for the big three infectious diseases (HIV/AIDS, malaria,
tuberculosis). However, given the increasing global importance of
dengue, the globalization of information and, perhaps, the advocacy
on global warming and its potential effect on the geographic
distribution of diseases, interest in den-gue has increased.
Advocacy and incentives for are still needed for scientists in
academic institutions to work on dengue, and to improve career
opportunities in the field. This could be achieved through
dedicated funds and grants available for applied research on
dengue. Such funds could be provided by public health driven
governmental and/or non-governmental organizations, or pri-vately
driven foundations such as the Pediatric Dengue Vaccine Initiative
(PDVI) which has dedicated dengue vaccine evaluation and access
programs.
Dengue research has not yet reached a critical mass in many
countries where the disease is endemic and ranks among the top five
public health concerns. The PDVI, in collaboration with the Pan
American Health Organization (PAHO) and WHO, recently organized
regional dengue research meetings assembling scientists from Asia
and the Americas. Such meetings help gather
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