Use of amplicon-6 vectors derived from human herpesvirus 6 for efficient expression of membrane-associated and -secreted proteins in T cells.

The composite amplicon-6 vectors, which are derived from human herpesvirus 6 (HHV-6), can target hematopoietic cells. In the presence of the respective helper viruses, the amplicons are replicated by the rolling circle mechanism, yielding defective genomes of overall size 135 to 150 kb, composed of multiple repeats of units, containing the viral DNA replication origin, packaging signals, and the selected transgene(s). We report the use of amplicon-6 vectors designed for transgene expression in T cells. The selected transgenes included the green fluorescent protein marker, the herpes simplex virus type 1 glycoprotein D (gD), and the gD gene deleted in the transmembrane region (gDsec). The vectors were tested after electroporation and passage in T cells with or without helper HHV-6A superinfections. The results were as follows. (i)The vectors could be passaged both as cell-associated and as cell-free secreted virions infectious to new cells. (ii)The intact gD accumulated at the cell surface, whereas the gDsec was dispersed at internal locations of the cells or was secreted into the medium. (iii)Analyses of amplicon-6-gD expression by flow cytometry have shown significant expression in cultures with reiterated amplicons and helper viruses. The vector has spread to >60% of the cells, and the efficiency of expression per cell increased 15-fold, most likely due to the presence of concatemeric amplicon repeats. Current studies are designed to test whether amplicon-6 vectors can be used for gene therapy in lymphocytes and whether amplicon-6 vectors expressed in T cells and dendritic cells can induce strong cellular and humoral immune responses.