Formulation and delivery of dermal DNA vaccines

Abstract

DNA vaccination is an appealing strategy of active vaccination, leading to the intracellular production of the encoding antigen which results in an efficient activation of an antigen specific immune response. Intradermal DNA tattooing was recently developed as a simple and robust method to induce antigen expression and T cell responses. This thesis focused on the pharmaceutical production and formulation, and the optimal delivery of DNA vaccines administered by DNA tattooing. First, a process is described for the production of naked DNA vaccines for clinical trials. The developed production process was reproducible, with a high quality, stability and yield of plasmid DNA. The manufactured melanoma DNA vaccine, pDERMATT (encoding Tetanus Toxin Fragment C and a MART-1 epitope), showed a robust T cell response in a humanized murine model vaccinated by DNA tattooing and is currently under clinical evaluation. Next, the effect of lipopolysaccharide (LPS), a common contamination, was assessed. The presence of LPS in DNA vaccines, applied via DNA tattooing, neither induced systemic toxicity nor influenced antigen expression in mice. LPS content was not associated with a statistically significant increase in T cell response. The development of an ex vivo human skin model that allows the longitudinal measurement of vaccine induced antigen expression in intact skin is described. This skin model demonstrated that the majority of cells transfected by DNA tattooing are epidermal keratinocytes, with only a small fraction (~1%) of antigen-positive epidermal Langerhans cells. Furthermore, we showed that an increase in DNA concentration, needle depth and tattoo time all significantly increase antigen expression, with DNA concentration forming the most critical variable. The transfection efficiency of DNA tattooing appeared to be extremely low, only 1 out of 5x106 to 5x109 copies of plasmid applied is taken up, transcribed and translated. To further increase this low transfection efficiency, we developed a nanoparticle formulation for the delivery of dermal DNA vaccines. It was shown that the shielding of the surface charge of a cationic nanoparticle (both lipoplexes and polyplexes) via PEGylation is essential to induce both antigen expression and immunogenicity. NonPEGylated particles carry a positive zeta potential and therefore likely bind to negatively charged tissue matrix macromolecules, preventing cellular binding and internalization. Subsequently, the effect of tattooing on plasmid DNA integrity (supercoiled (SC) content) was assessed. DNA tattooing only resulted in minor damage to pDNA ( <3% SC pDNA reduction) and only open circular (OC) pDNA formation. Surprisingly, antigen expression levels and immunogenicity upon DNA tattooing of SC or OC pDNA were equal in a murine model, whereas tattooing in ex vivo human skin resulted in a significant higher level of antigen expression for the SC form. DNA topology did not influence expression in human skin when formulated as PEGylated polyplexes. To further increase the transfection efficiency, we described the combination of DNA tattooing with electroporation. First, it was shown that tattooing immediately followed by electroporation (“electrotattooing”) can increase antigen expression levels in human skin by a factor of 3. Even more interestingly, electrotattooing of a polyplex formulation boosts antigen expression up to levels 20-fold higher compared to tattooing of naked DNA