High-risk Human Papilloma Virus (HPV) induced malignancies are an interesting target for immunotherapy as they invariably express the viral proteins E6 and E7 that allow specific recognition of... Show moreHigh-risk Human Papilloma Virus (HPV) induced malignancies are an interesting target for immunotherapy as they invariably express the viral proteins E6 and E7 that allow specific recognition of tumor cells without the risk of inducing autoimmunity. This thesis describes the preclinical development of pDNA vaccine candidates for the treatment of HPV16 induced malignancies. For this purpose gene-shuffled (SH) versions of HPV16 E6 and E7 were used to avoid the risk of transformation at the vaccination site. To improve the immunogenicity of E6SH and E7SH several optimizations strategies were evaluated. First it is shown that genetic fusion with Tetanus Toxin Fragment C (TTFC), results in a dramatic increase in immunogenicity. The resulting vaccine candidates TTFC-E6SH and TTFC-E7SH were also demonstrated to have lost their transforming potential. Secondly it is demonstrated that the mere addition of an endoplasmatic reticulum targeting sequence and a set of promiscuous CD4 helper epitopes results in an enormous increase of the immunogenicity of E6SH and E7SH. This design decreases the risk of raising competing carrier specific immune responses. Finally several nanoparticle based formulations of a model pDNA vaccine were evaluated and it is demonstrated that for in vivo applications it is essential to shield the cationic charge of such nanoparticles. Show less
