The mosquito-borne flavivirus, Dengue, is estimated to cause in each year 100 million cases of Dengue fever (DF), 500,000 cases of Dengue Hemorrhagic fever (DHF) and 25,000 deaths, with 2.5 billion people at risk [1]. Although a successful vaccine against the prototypical flavivirus, yellow fever (YF) virus, has been in use since the 1930s and vaccines to two other flaviviruses, Japanese encephalitis (JE) virus and tick-borne encephalitis (TBE) virus are currently available, there is as yet no Dengue vaccine approved for use [2].
Dengue virus has a typical flavivirus genome structure, as described in Figure 1A. The structural proteins, C, prM (M) and E, are involved in packaging, export and subsequent entry. The non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 include an RNA-directed RNA polymerase, and a protease function involved in cleaving certain positions of the long viral polyprotein which contains all the viral genes [3, 4].
The four serotypes of Dengue virus ("1" through "4") share approximately 60%–74% amino acid residue identity with one another in the E gene [5] and induce cross-reacting antibodies [6]. However, neutralizing antibodies to the structural proteins of one serotype of Dengue typically not only fail to provide protection against other serotypes, but appear to cause the enhanced replication of virus seen in Dengue hemorrhagic fever, which is generally seen upon reinfection by Dengue virus of a different serotype. This antibody-dependent enhancement of infection (ADE), which is believed to be mediated by enhancement of viral uptake by macrophages [7] complicates Dengue vaccine development, since an inadequate or modified immunogen may contribute to disease, rather than prevent infection [8].
Two strategies suggest themselves for circumventing the problems caused by cross reacting antibodies against the major structural proteins, prM and E. One strategy is to immunize with multiple strains of Dengue virus to elicit high affinity, neutralizing antibodies against the multiple Dengue serotypes. At least one vaccine to do this (using dengue vaccine candidates DEN-1 PDK13, DEN-2 PDK53, DEN-3 PGMK 30/F3, and DEN-4 PDK48) has been in clinical trials [9, 10]. A second strategy is to induce immunity only to viral proteins other than prM and E. Several studies have shown that the nonstructural glycoprotein NS1 can play an important role in protection against Dengue. Mice immunized with purified Dengue-2 NS1 protein injected intramuscularly and boosted after 3 days and two weeks were protected from developing lethal Dengue encephalitis upon subsequent challenge with Dengue 2 virus. [11]. Similarly, mice immunized with recombinant vaccinia virus expressing authentic NS1 [12] were protected against the development of Dengue-4 virus encephalitis when challenged by intracerebral injection. Inoculation of mice with specific combinations of MAbs directed against Dengue-2 NS1 [13] also protects against lethal virus encephalitis upon intracerebral Dengue-2 challenge. Other nonstructural proteins are also immunogenic and may participate in eliciting protection [14].
Towards the goal of devising a "live" vaccine based on only non-structural Dengue proteins, we have attempted to construct Dengue virus genomes from which the pre-M and E genes have been deleted. Upon introduction into a host's cells, these sub-genomic fragments should replicate intracellularly and support prolonged expression of Dengue non-structural proteins without producing the deleted structural proteins and without forming infectious virions. Sub-genomic replicons of several positive-strand RNA animal viruses have been reported, particularly yellow fever and Kunjin among the flaviviruses. These replicons, when introduced into host cells, replicate and make viral proteins for over 41 days [15], but cannot form infectious virions because they lack critical structural proteins. Effectively delivered to host cells in vivo, such replicons should efficiently induce immunologic reactions against the expressed proteins remaining in the sub-genomic construct. Here we describe the successful construction of two Dengue virus sub-genomic constructs which replicate in LLC-MK2 cells in tissue culture when transfected in as full length RNA. We also report that expression of Dengue virus proteins from at least one of these replicons can be supported by transfection of a DNA-based expression vector containing the replicon.