The successful induction of a T-cell-mediated tumor-protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. We achieved this by immunization with a tyrosine hydroxylase (mTH)-based DNA vaccine, enhanced with the posttranscriptional regulatory acting RNA element (WPRE), derived from woodchuck hepatitis virus in combination with an antibody-cytokine fusion protein (ch14.18-IL-2) that targets interleukin-2 (IL-2) to the tumor microenvironment. This DNA vaccine mTH-WPRE was carried by attenuated Salmonella typhimurium and applied by oral gavage in a mouse model of neuroblastoma. Mice immunized with the mTH-WPRE vaccine, and which additionally received a boost with suboptimal doses of ch14.18-IL-2, were completely protected against hepatic neuroblastoma metastases. In contrast, all controls presented with disseminated metastases. Both T-cell and natural killer (NK) cell-dependent mechanisms were involved in the induction of a systemic tumor-protective immunity. Thus, up-regulation of interferon-gamma (IFN-gamma) expression in CD8(+) T cells occurred only in those animals that received the mTH-WPRE vaccine plus the ch14.18-IL-2 boost. Up-regulation of this proinflammatory cytokine was not observed in mice immunized with mTH-WPRE vaccine alone. A role for NK cells was indicated by the complete abrogation of systemic tumor-protective immunity in all animals that were depleted of NK cells in vivo. Taken together, these data demonstrate that immunization with a posttranscriptionally enhanced DNA vaccine encoding the WPRE sequence, combined with a boost of the ch14.18-IL-2 fusion protein, completely protects against hepatic metastases in a murine model of neuroblastoma and therefore may lead to a new strategy for immunotherapy and prevention of metastatic neuroblastoma.