Introduction: Tyrosine hydroxylase (TH) is highly expressed in neuroblastoma (NB) and therefore a promising target for active immunity approaches. Interleukin-15 (IL-15) represents an attractive adjuvant for DNA vaccination due to its ability to induce cytotoxic but not regulatory T cells. Here, we report generation and characterization of a new bicistronic DNA vaccine encoding for TH and IL-15 for active immunotherapy against NB.
Methods and Results: The bicistronic mammalian vector system pIRES allows simultaneous expression of two genes of interest from the same bicistronic mRNA transcript due to an internal ribosome entry site (IRES) flanked by two multiple cloning sites (MCS). To generate the new DNA vaccine, a previously described DNA sequence encoding for TH combined with upstream Ubiquitin (Ub) was cloned into the first MSC of pIRES using standard molecular biology techniques. Ub ensures proteasomal degradation of TH in antigen-presenting cells and its MHC class I presentation to cytotoxic T lymphocytes (CTL) followed by their activation. Synthesis and insertion of murine splenocyte IL-15 cDNA-sequence into the second MSC of pIRES allows secretion of the bioactive cytokine into the micro milieu stimulating TH-specific CTL activation. In order to prove cytokine secretion in vitro, an IL-15 specific ELISA was performed using supernatants of CHO cells transfected with the TH and IL-15 containing plasmid. Correct insertion of both gene sequences TH and Il-15 was verified by gene-specific PCR as well as restriction- and sequence analysis. To evaluate anti-tumour immunity in vivo, a syngenic NB mouse model will be used. Thereby, A/J mice will be vaccinated with the new vaccine by oral application of plasmid-bearing attenuated Salmonella typhimurium SL7207, which are known as an effective transport vehicle for oral immunization.
Conclusion: We generated and partly characterized a new bicistronic DNA vaccine encoding for TH and IL-15 to induce a long lasting immunity against NB.