Recently, DNA tattooing was introduced as novel intradermal administration technique for plasmid DNA (pDNA) vaccines. The aim of this study was to determine if tattooing affects the integrity of pDNA (reduction in supercoiled (SC) content) and whether a change in pDNA topology would affect antigen expression and immune response. We show that 1.) in vitro tattooing of pDNA solutions results in minor damage to pDNA (<or=3% SC pDNA reduction) and only open circular (OC) pDNA formation, 2.) antigen expression and T-cell responses upon tattoo administration of SC and OC pDNA are equal in a murine model, 3.) SC pDNA gives a significantly higher antigen expression than OC and linear pDNA in ex vivo human skin, 4.) pDNA topology does not influence antigen expression when formulated as PEGylated polyplexes. We conclude that a 3% reduction in SC purity most likely will have little or no effect on clinical antigen expression and T-cell responses. For intradermal tattoo administration the ex vivo skin model might be more suitable than the standard murine model for distinguishing subtle alterations in antigen expression of clinical pDNA formulations. The results from this study enable justification of release and shelf-life specifications of pDNA products applied by this specific route of administration, as requested by the regulatory authorities (>or=80% SC).