Cytolytic Perforin as an Adjuvant to Enhance the Immunogenicity of DNA Vaccines

Shrestha, Ashish C.; Wijesundara, Danushka K.; Masavuli, Makutiro G.; Mekonnen, Zelalem A.; Gowans, Eric J.; Grubor-Bauk, Branka

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] . 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 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] .