Abstract
As part of a program to develop a Dengue virus vaccine which avoids the deleterious effects of antibody dependent enhancement (ADE) of infection mediated by antibodies to Dengue virus structural proteins, we have begun to investigate the possibility of designing Dengue vaccines based on non-structural proteins. Dengue constructs which lack major structural proteins replicate intracellularly in tissue culture. These replicons are capable of prolonged expression of Dengue virus non-structural proteins for at least seven days in culture. Dengue virus genomes lacking major structural proteins can, like other flaviviruses, replicate intracellularly and express virus non-structural proteins with minimal toxicity to host cells. These findings pave the way for the development of dengue virus replicons as a form of live, attenuated virus vaccine.
Highlights
As part of a program to develop a Dengue virus vaccine which avoids the deleterious effects of antibody dependent enhancement (ADE) of infection mediated by antibodies to Dengue virus structural proteins, we have begun to investigate the possibility of designing Dengue vaccines based on non-structural proteins
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]
Neutralizing antibodies to the structural proteins of one serotype of Dengue typically 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
Summary
We have demonstrated the prolonged expression of Dengue virus proteins from sub-genomic Dengue RNA fragments, lacking major structural genes, transfected into tissue culture cells. Dengue-2 virus cDNA cloned in the yeast shuttle vector pBR424, linearized by excision of a short Bam HI fragment was transfected into competent [20] S. cerevisiae YPH857 (kindly provided by Barry Falgout (CBER/ FDA), along with the appropriate PCR fragment spanning the desired deletion. Aliquots (12.5 ul) of the reaction mixtures, containing full length viral RNA, were used to transfect approximately 2 × 106 monkey LLC-MK2 cells in phosphate-buffered saline (PBS) by electroporation in a 0.4 cm gap electroporation cuvette. Cells were either plated directly on multiwell plates for harvest at short time periods (typically 48 hrs or less) or on tissue culture dishes for trypsinization and seeding onto multiwell plates on the day before final harvest for longer time periods (typically 8 days post transfection). Cells in some experiments were counterstained with 0.02% Evans Blue
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