Previous results showed that cells infected with recombinant vaccinia virus containing cloned dengue DNA sequences produced what appeared to be authentic dengue Pre M, E, and NS1 glycoproteins. However, most animals inoculated with this recombinant virus failed to develop immune responses. In order to improve the immunogenicity of recombinant DNA produced dengue proteins, experiments were initiated to produce individual dengue antigens which were expressed on the cell surface or secreted extracellularly. The hemagglutinin (HA) of influenza virus was chosen to provide the N-terminal signal as well as the C-terminal anchor sequences that are required for intracellular protein transport. DNA sequences coding for the E glycoprotein were constructed by joining the Sal I/Acc I cleaved dengue DNA fragment to chemically synthesized oligomers. To prepare expression vector DNA, internal sequences of HA were removed by XbaI and XhoI digest and subsequently by Bal 31 for further extension from the cleavage site. This collection of HA deletions will be used for insertion of the E DNA sequences and expression of the recombinant DNA using the SV40 system. Recombinant SV40 virus producing the chimeric HA-E protein on the cell surface or secreted extracellularly will be identified by immunofluorescence assay. Finally, the DNA sequences expressing the chimeric protein will be isolated and used for construction of live recombinant vaccinia virus.
