Defined Flanking Spacers and Enhanced Proteolysis Is Essential for Eradication of Established Tumors by an Epitope String DNA Vaccine

Abstract

Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 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 challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.

The efficacy of a DNA vaccine containing inserted and replicated regions of the E7 gene for treatment of HPV-16 induced tumors

“…The Helios gene gun system (Bio-Rad, Hercules, CA) was used for intradermal delivery of vaccine and control genes as previously described [7]. Bullets containing 2 μg of DNA/shot were generated according the manufacturer's protocols.…”

“…The nucleotide sequence was synthesized by Entelechon (Entelechon GmbH, St. Veit-Weg 2, 93051 Regensburg) and was cloned into a PCR4 topo vector and sub-cloned into the APL023 expression vector [7]. In the a region, amino acids 21, 24, and 26 were modified from DLYCYEQ to GLYGYGQ to reduce transformation activity and enhance antigenicity [17,18].…”

“…Several different approaches have been examined including the attachment of ubiquitin to DNA constructs [6,7], targeting of tumor antigens to centrosomal compartments to enhance MHC class I presentation [8], the attachment to constructs of calreticulin to target HPV antigens to the MHC I pathway [9], optimization of codon usage for enhanced expression and presentation of HPV oncogenes [6,10,11] , utilization of modified genes to decrease the transformation capacity [12][13][14] or enhancement of intercellular spreading in order to increase the number of cells expressing antigen [15]. All of these approaches have demonstrated increased antigen presentation and superior tumor eradication in mouse models.…”

“…In the c region two zinc finger binding motifs were disrupted by a split between amino acids 60 and 61 and modifications of C61 to G61 and C94 to G94. All three regions, a, b, c were replicated to enhance expression, and H-2Db and HLA-A*0201 epitopes were added downstream of the aabbcc construct with Ala-Ala-Tyr (AAY) as an optimal spacer for proteosomal cleavage [7]. In addition to further enhancing expression, addition of these epitopes provided unique primer sites at the 3' end of the construct that were necessary for cloning.…”

“…Lastly, the codons in the E7IR construct were optimized for expression in mice. The construct was cloned into the multiple cloning site of the APL023 expression vector [7] utilizing Bam H I and Not I restriction enzymes. …”