Project Lead(s): Bunpote Siridechadilok
Dengue is a major threat to public health in most tropical and subtropical countries, with an estimated 100 million infections annually.
Despite intensive work, there is still no vaccine to provide protection against dengue infection, which is caused by one of four dengue virus serotypes.
The development of a dengue vaccine has unique challenges as the four dengue serotypes circulate globally, and infection with one dengue serotype confers life-long protection against re-infection with the same serotype but only short-term protection against the other three serotypes.
Moreover, dengue is unique in that sequential infections with different serotypes increase the risk of developing severe and potentially lethal disease.
A safe and effective dengue vaccine must be tetravalent and induce strong and long-lived protection against all four serotypes simultaneously, in order to avoid the risk of sensitizing the vaccine recipient to severe disease.
Conventional virus-construction techniques can produce only a small number of virus mutants at a time and, with a small mutant pool available for analysis, the chance of finding target vaccine candidates is low.
The project team hoped to combine a novel virus-construction tool with existing high-throughput assays to effectively search for new vaccine candidates for dengue.
The aim was to create a virus-mutant pool at least 100,000 times larger than the pool derived by traditional techniques.
Specifically, the researchers wanted to derive dengue vaccine candidates through functional high-throughput screening of the dengue-virus (DENV) library for the mutants that are sensitized toward human STAT2 gene.
Human STAT2 is a master transcription regulator that propagates antiviral interferon response and is antagonized by natural DENV. Thus, such mutants could serve as attenuated DENV strains that could be further developed into vaccines.
The project failed in obtaining DENV mutants that were sensitized specifically toward the over-expression of STAT2 (and not of other anti- dengue genes).
Several technical difficulties were encountered in setting up all the components used in the screening system, and in dealing with cost and resourcing issues.
The project team was able to set up the reporter virus and the stable cell that over-expresses STAT2. The cell and the reporter system were able to replicate the suppression of dengue virus upon over-expression of STAT2. During this project, the team was also able to reduce the cost of DNA assembly to support large-scale DNA assembly.
The team believes there are areas of future research to be explored, including:
· Characterization of the isolated mutants to test their suitability as vaccine candidates for trial.
· Analysis of identified mutations and testing the identified mutations in other serotypes.
· The application of the technique to isolate DENV variants that are attenuated against other antiviral genes identified in previous studies.
· The application of the technique to derive DENV that is resistant to mouse interferon system.