expressing a shRNA able to silence those molecules that negatively control the immune response.35 This shRNA-mediated interfer- ence with regulatory mechanisms only concerns plasmid- transfected antigen-presenting cells. When the ability of these transfected cells to induce an efficient immune response is disturbed by a tumor, neutralization of tumor-borne regulatory factors may result in an efficient presentation of oncoantigen peptides.36 The Sequence Coding for the Antigen Fine modifications of the sequence coding for the antigen protein lead to major differences in protein processing and immunogeni- city.5 Addition of a signal peptide or an ubiquitine signal to the N-terminus of the antigen sends the encoded protein toward the extracellular microenvironment through the endoplasmic reticu- lum or toward the proteasome for processing and presentation by MHC class I glycoproteins,37 whereas it goes to the plasma membrane when idrophobic sequences are added to the C- terminus. Addition of a lysosomal targeting signal drives its presentation by MHC class II glycoproteins.38 The adaptive response elicited by the antigen can be “helped” by inserting additional sequences and obtaining an encoded protein fusing the antigen with these sequences.5 The fusion proteins containing tumor antigens and epitopes of the tetanus toxin engage T helper cells induced by a previous anti-tetanus immunization in the response against the tumor antigen. By exploiting receptors of different kind, the fusion antigen can be selectively targeted toward antigen presenting cells to empower them to in vivo priming of CD8+ T cells. When mice were electroporated with a plasmid encoding amino acid 364–391 of human ErbB-2 fused with the Fc domain of a human IgG1 (Fc- ErbB-2364–391), the better CD8 T cell-mediated response toward human ErbB-2 epitopes was paralleled by a stronger protection against a challenge of human ErbB-2+ mammary cancer cells.39 Another way we explored to get a more selective delivery of tumor antigens is to generate a plasmid coding for the antigen fused with the extracellular domain of CTLA-4, a receptor binding to the co-stimulatory B7 (CD86) molecules on DC. Following the injection in mice of a plasmid coding for the NH- terminal 222-amino acids of human ErbB-2 fused with the extracellular domain of human CTLA-4 we observed an enhanced anti-human ErbB-2 humoral and T cell mediated response and protection against a challenge with mouse tumor cells expressing human ErbB-2. A similar plasmid encoding a fusion CTLA-4- ErbB-2 mouse protein markedly delayed the onset of mammary carcinomas in BALB-neuT mice.40 We also searched for the smallest rat ErbB-2 epitope still able to elicit a protective response. Our starting point was the observation that RRTErbB-2 plasmid elicits a similar or even better response than the one encoding the full length rat ErbB-2.11 Then, we produced seven plasmids encoding the transmembrane domain associated with decreasing fragments of the extracellular domain.41 When these plasmids were electroporated in wild-type BALB/c mice, the intensity of the response and protection against a challenge of rat ErbB-2+ murine cells decreased with the shortening of the ErbB-2 fragment encoded. Surprisingly, immunization with the RRTErbB-2311–689 plasmid encoding a fragment lacking the first 311 amino acids of the ErbB-2 extracellular domain still afforded full protection. In BALB-neuT mice electroporation of RRTErbB-2311–689 induces a protection comparable to that elicited by RRTErbB-2. Its location in the transfected cell and the conformation acquired by the short fragment encoded probably allows a favorable exposition of critical ErbB-2 epitopes.41 One of the problems linked to the use of oncoantigens as a target is that they are self-tolerated molecules. Therefore, a vaccine should be able to induce an autoimmune response overcoming central and peripheral tolerance to self.42,43 Both the induction and the persistence of an immune response are markedly hindered by natural Treg cells that expand in response to the overexpression of self oncoantigens by the tumor. The “regulatory memory” constantly tuning down the immune response to self antigens44 makes the induction of an effective response to oncoantigens difficult, and make it necessary to continuously boost the vaccination to maintain its efficacy.45 Vaccination with xenogeneic ortholog antigens is an effective way to break self tolerance. Heterologous antigens activate both bystander anergic B and T cells and prime T cells. However, the cross-reactive response to self antigens may be of low intensity and affinity.46 When wild-type mice and mice transgenic and tolerant for the rat or the human ErbB-2 are immunized with plasmids encoding the extracellular and transmembrane domains of the fully human (HuHuTErbB-2) or fully rat ErbB-2 (RRTErbB-2), the response shows a consummate specificity to the rat or human ErbB-2 ortholog used as antigen but a poor cross-reactivity toward the ortholog expressed by the mouse and its tumors (Fig. 2). This “orthologue restriction” corresponds to a similar restriction in the ability to inhibit the growth of tumors expressing the human or rat ErbB-2 ortholog.22 To overcome it we immunized mice with plasmids coding for ErbB-2 proteins composed in part by rat and by human sequences. The homologous moiety guarantees the specificity of the response while the hetereologous moiety ensures better overcoming of tolerance (Fig. 2). Vaccination of wild-type mice and mice transgenic for the rat or the human ErbB-2 with these chimeric plasmids elicits a stronger and more cross-reactive response and a better protection than fully human or fully rat plasmids against carcinomas overexpressing either rat or human ErbB-2.22,45,47 Oncoantigens, Why? Tumor’s genetic instability often thwarts the immune attack because it results in the selection of clones that do no longer express the target antigen or express it in a way that it cannot be perceived by T cells.48,49 A conceptual quantum jump in planning new generation vaccines is to target the vaccine against oncoantigens since these are molecules not (easily) disposable during tumor progression nor are they easy to hide because of their indispensable role. Oncoantigen-loss variants can occur, but they will get a crippled tumorigenic potential and negative selection.50 Otherwise, the function of the targeted oncoantigen www.landesbioscience.com OncoImmunology 319