Figure 1. Cellular events following intramuscular electroporation of 50 mg of plasmid in 20 ml of saline through Cliniporator (Igea, Carpi, Italy). Insertion of the electroporator needles into the quadriceps muscle of the mouse and delivery of two low voltage pulses of 150V of 25 ms with a 300 ms interval (A). One-6 h later the damaged myofibers and cell debris (dotted yellow) are surrounded by polymorphonuclear (N) and mononuclear leukocytes (MW) (B). One or two days later mature and differentiated tissue macrophages (MW) and dendritic cells (DC) progressively become prominent among inflammatory cells infiltrating the numerous necrotic myofibers (C). On the third-fourth day from the electroporation, intact and regenerating muscular fibers (pale red) are overexpressing the protein encoded by the plasmid (green triangles), while the area is being infiltrated by B (B), T (T) and dendritic cells (DC). Interestingly, CD11b+ macrophages and CD11c+ DC and, later, CD4+ T cells are often in direct contact with antigen expressing muscle cells or antigen expressing fragments and each other (D). of particular reaction mechanisms. We have shown that the nonapeptide VQGEESNDK corresponding to the 163–171 amino acids of the human IL1β retains several of the immunostimulatory properties while it is free from the potent proinflammatory properties of the entire molecule and increases the immunogenicity of poorly immunogenic tumors.30 When BALB-neuT mice were immunized with RRTErbB-2 plasmids in which the sequence of this nonapeptide was inserted, a higher anti-rat ErbB2 antibody response accompanied by a massive lymphocyte infiltration of the mammary lesions went along with a stronger protection against mammary tumors.31 One of the most effective non-DNA vaccines against ErbB-2, the “Triplex ” vaccine, is based on mammary carcinoma cells expressing both the rat ErbB-2 protein and allogeneic class I major histocompatibility complex (MHC) glycoproteins administered in combination with recombinant IL-12.32 By replacing the recombinant cytokine with cytokine gene-engineered cells, it was possible to compare the efficacy of IFNc, IL-2, IL-12, and IL-15. In cancer prone ErbB-2 transgenic mice IL-12 was vastly superior to other cytokines.33 The translation of the Triplex design from cell-based to a DNA vaccine, using a mixture of three plasmids: RRTErbb2, a plasmid encoding full length H-2Dq MHC gene and another plasmid encoding mouse IL-12, showed that cell-based and the DNA Triplex vaccines were equally effective.34 However, in most instances vaccination with the RRTErbb2 plasmid alone elicited a protection similar to that obtained with the Triplex plasmids. This finding was surprising since our previous experience with cell-based vaccines had shown that the Triplex design yielded vastly superior protection from tumor onset in comparison to any subset of its components.32 Our results show that the strong immunity elicited by the RRTErbB-2 plasmid, already helped by embedded CpG sequences and by electropora- tion, is less dependent than analogous cell vaccines on the adjuvant activity of cytokines. Lastly, construction of more sophisticated plasmids results in the induction of better immune responses by simultaneously relieving from suppression. Plasmids can thus be endowed with two expression cassettes, one coding for the antigen, the other 318 OncoImmunology Volume 1 Issue 3