Superiority of ear skin for DNA immunization in mouse tumor models

Abstract

DNA vaccination can induce antibodies, helper T cell responses, CTL responses, and protective immunity in various animal models for infectious diseases and cancers. However, naked DNA immunization is still inefficient in large animals and human. I demonstrate in this thesis: i) that the site of DNA vaccine application is important; ii) that a viral DNA sequence can augment anti-tumor effects; iii) that electroporation improves anti-tumor immunity; and iv) that dendritic cells are essential and sufficient for antigen presentation in ear pinna DNA immunization. Intra-ear pinna (ie) DNA injection led to earlier and stronger antigen expression compared to intradermal injection at the flank skin (id). The ie site was superior to the id site also with regard to induction of humoral and cellular immune responses. To improve the anti-tumor effect of the DNA vaccines, an immunostimulating sequence coding for hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) was introduced as an adjuvant. HN expression in cells was demonstrated to induce IFN-α production, lymphocyte binding activity as well as anti-tumor activity. By combining this adjuvant with ie TAA (tumor associated antigen) DNA immunization, prophylactic and therapeutic anti-tumor immunity was improved in mouse tumor models. The tumor lines expressed either a surrogate tumor antigen beta-galactosidase (β-gal), or a TAA, human epithelial cell adhesion molecule (EpCAM). Improvements of the anti-tumor activity might be due to the observed increase of Th1 responses, anti-tumor CTL activity and innate immune reactivity, as well as due to down-regulated suppressive factors such as TGF-β and level of myeloid derived suppressor cells (MDSCs). To further improve the ie DNA immunization strategy, it was combined with electroporation (EP). Such DNA EP led to clear-cut improvements of humoral and cellular immune responses when applied ie. The effects in the ear pinna were superior to id DNA EP. In both prophylactic (β-gal as a TAA) and therapeutic (human EpCAM as a TAA) tumor models, DNA EP was demonstrated to increase anti-tumor activity significantly compared to DNA immunization without EP. I was able to identify a short DC-specific CD11c promoter sequence of 700-bp. Upon introduction into the DNA vaccine, such vector was found to induce similar anti-tumor immunity as a DNA vector driven by the CMV promoter although the latter led to much stronger antigen expression. This observation suggests that DCs are sufficient for antigen presentation of ear pinna DNA immunization. Thus, DNA vaccines encoding xenogeneic TAAs were particularly effective when applied to the ear pinna and induced protective and therapeutic anti-tumor immunity in mouse tumor models. The combination with HN as an adjuvant or with electroporation further augmented the anti-tumor effects. Studies on the mechanisms revealed that DCs in the ear pinna are essential for the immunization effect.