CRYPTIC OPEN READING FRAMES IN PLASMID VECTOR BACKBONE SEQUENCES CAN PROVIDE HIGHLY IMMUNOGENIC CYTOTOXIC T-LYMPHOCYTE EPITOPES

Murine tumor cells obtained through transfection of expression plasmid s carrying activated cellular and/or viral oncogenes constitute formid able tools for immunological tumor research. As reported previously, m ouse embryo cells of C57BL/6 origin, transformed by mutated p53 or hum an papilloma virus type 16 (HPV16), present, at their surface, MHC-bou nd peptides that are derived from the p53 and the HPV16 E7 oncoprotein s, respectively, which can serve as a target for a highly effective an titumor T-cell response. Here, we describe the identification, through molecular cloning, of an additional, highly immunodominant peptide th at is presented by the aforementioned HPV16- and p53-transformed cells , This peptide is encoded by a cryptic open reading frame in the backb one sequences of the plasmids that had been used to generate these cel ls. Considerable amounts of transcripts encompassing this open reading frame were detected in the cells concerned. These transcripts were th e result of the bidirectional nature of the retroviral long terminal r epeat (LTR) present in the expression plasmids used for transfection, which resulted in transcription of the gene of interest, as well as in transcription of the vector sequences positioned at the other side of the LTR, Due to this mechanism, all tumor cells harboring LTR-driven expression plasmids expressed the highly immunogenic peptide, whereas cells containing plasmids driven by more unidirectional promoters exhi bited lower levels of this peptide. LTR-driven expression plasmids wer e also shown to encode this peptide epitope when used for DNA vaccinat ion, as mice vaccinated with such a plasmid developed a CTL response a gainst this peptide. Our data show that awareness of plasmid backbone- derived epitopes is of crucial importance for the correct interpretati on of preclinical experiments and for the design of DNA vaccines.