THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2005; 7: 613–620. Published online 6 December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.702 Altered intracellular sorting signals do not influence the efficacy of genetic melanoma vaccines incorporating helper determinants in mice J¨ urgen Br¨ uck 1† Julia Steitz 1† Dennis Strand 2 Thomas T¨ uting 1 * 1 Laboratory of Experimental Dermatology, Department of Dermatology, University of Bonn, Germany 2 Department of Internal Medicine I, University of Mainz, Germany *Correspondence to: Thomas T¨ uting, Laboratory of Experimental Dermatology, Department of Dermatology, Rheinische Friedrich Wilhelm University, Sigmund Freud Str. 25, 53105 Bonn, Germany. E-mail: thomas.tueting@ukb.uni-bonn.de † These authors contributed equally to this work. Received: 25 July 2004 Revised: 22 September 2004 Accepted: 23 September 2004 Abstract Background A genetic melanoma vaccine consisting of cDNA encoding the model self-antigen tyrosinase-related protein 2 (TRP2) fused in-frame to the immunogenic enhanced green fluorescent protein (EGFP) was able to break immune tolerance and stimulate CD8 + T cells in vivo. In the present study we investigated whether alteration of the intracellular antigen localization as a result of the linkage with immune-enhancing helper proteins affects the resulting immune response. Methods Expression plasmids and recombinant adenoviruses were con- structed encoding various fusion proteins with different intracellular sorting signals which direct the antigen to the cytosol, the endoplasmic reticulum or the endosomal compartments. Genetic immunization of C57BL/6 mice was performed with all constructs using particle-bombardment of the skin and injection of recombinant adenoviruses. The resulting immune response was analyzed using ELISPOT and tumor rejection assays. Results Induction of TRP2-specific CD8 + T cells in vivo and autoimmune- mediated destruction of melanocytes in the bombarded area of the skin were observed with all constructs expressing fusion proteins between TRP2 and EGFP. Importantly, injections of the different recombinant adenoviruses all mediated protective immunity against transplanted B16 melanoma cells. Conclusions Altered intracellular sorting signals do not significantly influence the efficacy of genetic melanoma vaccines incorporating helper determinants in our model system. These results further support the concept that linkage of immunogenic helper sequences can be successfully applied for antigen-specific immunotherapy of melanoma and provide a scientific basis for the translation of this strategy in future clinical applications. Copyright  2004 John Wiley & Sons, Ltd. Keywords tumor immunology; genetic immunization; melanoma; T helper cells; TRP2 Introduction Linkage of weakly immunogenic tumor antigens with immune-enhancing helper proteins has recently been identified as an effective strategy to promote the induction of antigen-specific tumor immunity in mice using various experimental models [1,2]. We reported that this strategy can be successfully applied for melanoma vaccination [3]. Using the melanosomal Copyright  2004 John Wiley & Sons, Ltd.