CONSTRUCTION OF ADENOVIRAL AND RETROVIRAL VECTORS COEXPRESSING THE GENES ENCODING THE HEPATITIS-B SURFACE-ANTIGEN AND B7-1 PROTEIN

Recombinant retroviral (re-Rv) and adenoviral (re-Ad) vectors for deli very of two foreign genes were constructed, using the internal ribosom al entry site (IRES) of encephalomyocarditis virus (EMCV) which mediat es initiation of cap-independent translation. The first gene encoded t he hepatitis B surface antigen (HBsAg) and the second encoded human or murine B7-1 molecule, a cell surface protein which is a costimulator for T cell activation. The EMCV IRES sequence was placed between the f irst and second coding sequences to form a dicistronic DNA fragment. I n Rv vectors, the dicistronic fragment was inserted between the 5' lon g terminal repeat (LTR) and an internal promoter for the neomycin (neo ) gene, so that the transcription initiated from the 5' LTR would gene rate a dicistronic mRNA for the HBsAg and B7-1 molecules. For Ad vecto rs, the dicistronic fragment was inserted between a cytomegalovirus pr omoter and a polyA signal to form a transcription cassette. This trans cription cassette was inserted into the early region 1 of Ad5 genome t o form a replication-defective re-Ad vector, or into early region 3 to form replication-competent vectors. Human cell line A549 infected wit h the re-Rv vectors or with the re-Ad vectors synthesized and secreted HBsAg at comparable levels, while the B7-1 molecules were detected at the surface of the infected cells, indicating both foreign genes carr ied by the Rv and Ad vectors were expressed efficiently.