Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine

Loss of immunogenic epitopes by tumors has urged the development of vaccine s against multiple epitopes. Recombinant DNA technologies have opened the p ossibility to develop multiepitope vaccines in a relatively rapid and effic ient way. We have constructed four naked DNA-based multiepitope vaccines, c ontaining CTL, Th cell, and B cell epitopes of the human papillomavirus typ e 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor ch allenge. The addition of spacers between the epitopes was crucial for the e pitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When t ested for therapeutic potential, only the epitope construct with defined sp acers significantly reduced the size of established tumors, but failed to i nduce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-in duced T cell-mediated eradication of 100% of 7-day established tumors in mi ce. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccin es against established tumors will be of importance for the further develop ment of multiepitope DNA vaccines toward clinical application.