DEVELOPMENT OF VECTORS FOR EXPRESSION OF VIRAL ENVELOPE GLYCOPROTEIN ANTIGENS

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Development of dengue (DEN) virus candidate vaccine antigens by genetic engineering has focused on modification of viral vectors to enhance protein expression and antigen secreation. Sindbis (SIN) virus replicons, SIN virus biphasic viruses and the T7 vaccinia recombinant systems were genetically engineered to express DEN virus premembrane (prM) and envelope (E) protein antigens. High level expression of DEN virus antigens was observed in cells infected with these vectors containing the DEN virus prM and E genes. However, the majority of the proteins remained intracellular suggesting that the expressed proteins were improperly processed or folded preventing extracellular secreation. To improve intracellular processing and enhance secreation of the proteins from infected cells it was essential to include in the cloned DEN protein gene cassette, genes encoding the last 15 amino acids at the carboxyl end of the capsid protein in addition to the basic amino acids at the prM/E cleavage site. DEN virus genes encoded into the bicistronic SIN virus were unstable. To further enhance expression of genes by the SIN virus replicon, the vector was genetically engineered to facilitate in vivo transcription of the subgenomic RNA by the addition of a RNA transcription start site under control of a RSV eukaryotic promoter and attachment of a deltaribozyme at the end of the viral 3'-NTR encoding the DEN insert. These changes simplified the transfection process and improved gene expression. The recombinant vaccinia vector pTM-1 into which DEN virus prM and E gene were cloned expressed high levels of DEN virus protein in both the transient system and recombinant vaccinia virus carrying the T7 polymerase. To facilitate selection of recombinant vaccinia viruses encoding viral genes, the neomycin resistance gene was engineered into the vaccinia vector under control of the 7.5 promoter outside of the TK-region used for homologous recombinantion. Studies to enhance levels of DEN virus prM and E protein produced and facilitate secreation of the antigens are currently in progress.