The initial goal in this project was to express both protective antigens of dengue virus, i.e., the E glycoprotein and the NS1 nonstructural glycoprotein from cloned dengue cDNA using vaccinia virus as a vector. Because these and other dengue proteins are presumably proteolytically cleaved from their polyprotein precursor, the 5' region of the dengue genomic cDNA that codes for all the structural proteins, (C, (PreM) M, and E) as well as the first two downstream nonstructural proteins, NS1 and NS2a, was inserted into vaccinia virus. In this construct the dengue cDNA insert is under the control of the P7.5 early-late vaccinia promoter, the vector contains the E. coli Beta-galactosidase gene under the control of a separate (P11) vaccinia promoter and both chimeric genes are flanked by vaccinia thymidine kinase (TK) sequences. Initially, dengue viral antigens were detected by indirect immunofluorescence in CV-1 cells infected with the vaccinia-dengue recombinant. Specific identification was made using a dengue type 4 virus E glycoprotein monoclone or a polyvalent dengue type 4 virus antiserum prepared in mice. Vaccinia-dengue recombinant infected cells were then labeled with 35S-methionine and the cell lysate was processed for immunoprecipitation in order to determine which dengue-specific viral products were expressed. SDS-polyacrylamide electrophoresis indicated that the dengue E glycoprotein monoclonal antibody precipitated a protein of approximately 50-55 Kilo daltons (Kd) molecular weight which is the expected size of the E glycoprotein. Similarly, a dengue type 2 virus NS1 antiserum precipitated a 35-38 Kd protein; this size is consistent with the molecular weight of the NS1 protein. These observations suggest that both glycoproteins were synthesized and specifically processed in a manner similar to that of authentic dengue proteins in virus infected cells. Currently, cotton rats are being immunized with the vaccinia-dengue recombinant.
