**3. Cytolytic DNA Vaccines**

Cytolytic DNA vaccines are based on a bicistronic DNA plasmid constructed on a pVax backbone (Invitrogen) with a Cytomegalovirus (CMV) promoter and a Simian Virus (SV40) promoter [55]. The gene encoding the protein of interest as an immunogen is inserted downstream of the CMV promoter and the gene encoding a truncated version of mouse PRF downstream of the SV40 promoter (Figure 1). The SV40 is a weaker promoter compared to CMV and has been shown to result in 10-fold lower protein expression in transfected HEK293T cells in vitro [55,95].

**Figure 1.** Schematic diagram of cytolytic bicistronic DNA plasmid (rDNA-PRF) with two different promoters and encoding protein of interest (immunogen) and truncated perforin.

The PRF gene was modified to express a truncated version of PRF (~60KDa) lacking the final 12 amino acid residues of the C terminus (unstructured region of PRF) [56,57,94] in order for PRF to become cytolytic [96]. The final 12 C-terminal amino acids are required to export PRF protein from the endoplasmic reticulum (ER) to the Golgi, from where glycosylated PRF is then transported to secretory granules [96]. Removal of the C-terminal abrogates the export of PRF from the ER and its subsequent accumulation in the ER is cytotoxic to the host cell [96,97].
