Plasmids encoding protein aggregation domains act as molecular adjuvants for DNA vaccines

Background: DNA vaccines provide high tolerability and safety but commonly suffer from suboptimal immunogenicity. We previously reported that a plasmid vector (pATRex), encoding the DNA sequence for the von Willebrand I/A domain of the tumor endothelial marker-8 (TEM8) when given in combination with plasmid-encoded tumor antigens acted as a powerful molecular adjuvant enhancing immunity against breast and melanoma tumors. Aims: In the present study we ad- dressed two unsolved issues; would the adjuvant action of pATRex extend to a DNA vaccine against infectious disease and, if so, what is the mechanistic basis for pATRex adjuvant action? Results: Here we show in a murine malaria vaccine model that co-administration of pATRex potentiates antibody production elicited by an intramuscular injection of plasmid encoding Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5). pATRex enhanced the B-cell response and induced increased IgG1 production consistent with TH2 polarization of the DNA vaccine response. To explore the mechanism of adjuvant ac- tion, cells were transfected in vitro with pATRex and this resulted in formation of insoluble intracellular aggregates and apop- totic cell death. Using a structural modeling approach we identified a short peptide sequence (α3-β4) within ATRex responsible for protein aggregation and confirmed that transfection of cells with plasmid encoding this self-assembling peptide similarly triggered intracellular aggregates, caspase activation and cell death. Conclusion: Plasmids encoding aggregation-promoting domains induce formation of insoluble intracellular aggregates that trigger caspase activation and apoptotic cell death leading to activation of the innate immune system thereby acting as genetic adjuvants.