DEN viruses, of which there exist four distinct serotypes (DEN-1, -2, -3 and -4), belonging to the family Flaviviridae, are transmitted to humans by the mosquito Aedes aegypti.
Infection with DEN viruses poses a significant public health threat to
~40% of the global population. DEN virus infections, of which ~100
million cases occur annually, produce symptoms ranging from mild fever
to severe hemorrhagic, potentially fatal, fever [1-3].
The pathogenesis of severe disease is not very well understood. Though
several factors such as virulence of the infecting virus, age and
genetic predisposition of the patient are implicated [4,5], the most important factor is held to be sequential infection by different serotypes in areas of endemicity [1-3].
Antibodies elicited by a given DEN virus serotype, which confer
lifelong homologous immunity to that serotype, can cross-react with,
but not protect against the remaining serotypes. These in fact have the
potential to exacerbate disease severity through antibody dependent
enhancement (ADE), during secondary infection with a different serotype
Additionally, pre-existing memory T cell populations with lower avidity
for the secondary serotype may be responsible for sub-optimal viral
clearance and enhanced pro-inflammatory responses .
Therefore, an ideal dengue vaccine should be 'tetravalent', capable of
affording protection against all four DEN virus serotypes [9,10].
In an effort to develop tetravalent dengue vaccines, several
investigators are currently exploring plasmid DNA-based experimental
vaccines in mice [11-13] and monkeys . Simultaneously, some groups are also investigating recombinant protein-based monovalent vaccine candidates [15,16]. Of the numerous DEN vaccine approaches being explored, live attenuated [17-23] and yellow fever vaccine vector YF17D-based chimeric [24-27] vaccines are in advanced stages of development [10,28]. Vector backbones based on attenuated DEN viruses are also being used to develop intertypic chimeric vaccines [29,30].
Uniformly, all these vaccine candidates are 'monovalent' in that they
are specific to a single DEN virus serotype. All these strategies rely
on physically mixing the four monovalent vaccines to generate empirical
'tetravalent' formulations [reviewed in [31,32]].
It is becoming increasingly apparent that these formulations have the
potential to elicit a skewed immune response, predominantly to a single
This has been attributed to 'viral interference', a poorly understood
phenomenon, associated with increased replication of one of the
monovalent vaccine viruses in the tetravalent formulation [reviewed in [31,32]]. This has necessitated the testing of multiple, empirical tetravalent formulations [20,22,27,30]. Recently, Zhou and Deem have suggested that injecting the monovalent vaccines at separate sites may alleviate this problem .
The objective of this work is to seek proof-of-concept for the
hypothesis that a single vaccine vector targeting multiple DEN virus
serotypes has the potential to bypass the risk of viral interference
inherent in the currently prevalent approach of mixing 'monovalent'
vaccines. For this purpose, we have developed a rAd vector encoding a
novel 'bivalent' antigen, based on EDIII of DEN-2 and DEN-4 viruses.
The choice of EDIII was based on several reasons. First, EDIII has
emerged as the most important domain from a vaccine perspective. It has
been shown that the host cell surface receptor-binding motif of the E
protein maps to EDIII ; further serotype-specific multiple neutralizing epitopes have been localized to this domain [35-37].
Importantly, EDIII has only a very low intrinsic potential for
eliciting cross-reactive antibodies against heterologous DEN virus
serotypes [38,39], implicated in severe pathogenesis. Second, DEN-2 virus EDIII protein (EDIII-2) can block DEN-2 virus infectivity [40,41]. Third, several groups [13,15,16,38,39] including ours [42,43]
have shown that EDIII-encoding gene-based vaccine candidates and
recombinant EDIII fusion proteins elicit neutralizing antibodies.
In this paper, we describe the construction and characterization of
a bivalent rAd vector encoding a chimeric antigen created by linking
EDIIIs of DEN virus serotypes 2 and 4 (EDIII-4/2 antigen), and show
that it elicits comparable levels of virus-neutralizing antibodies and
T cell responses specific to these two serotypes.