In Vivo Electroporation: The DNA Vaccinologist’s Dirty Little Secret There are several ways of delivering a naked plasmid. Its intramuscular injection is enough to elicit an immune response in experimental rodents and humans.8,9 The response is enhanced by the manipulation of the muscle through substances toxic for muscle cells10 or its surgical exposure.11 The plasmid can be enclosed in cationic liposomes to protect it against nucleases12 or shot into the dermis after coating it on gold particles by exploiting a “gene gun”13 or jet-injectors.14 Lastly, a rapid immune response follows tattooing of a plasmid into the skin.15 Just as adjuvants are the “immunologist’s dirty little secret”16 to increase the efficacy of a vaccine, electroporation is the dirty little secret that enhances the efficacy of DNA vaccines: (A) by facilitating plasmid entry into target cells; (B) by acting as an adjuvant that induces local tissue damage eventually attracting an inflammatory infiltrate. Delivery of short electrical pulses to the injection site causes temporary permeabilization of the cell membrane. Entry of the plasmid into the cell is facilitated and inter-individual variability of the response is minimized.17,18 Sophisticated electroporators have rendered plasmid electroporation a common way of securing efficient DNA immunization in animals19 and patients.20 Electroporators deliver pulses at a voltage that may be fixed or regulated to take account of tissue resistance. The number and kind of electric pulses, their voltage and their delivery through plaques applied on the skin or by needles determine the kind and intensity of the response elicited. In part this is due to the efficacy of the penetration of the plasmid into the cell. On the other hand, the kind of injury provoked by both needle insertion and electric pulses play a role in deciding the form and extent of cell death and the release of necrotic debris or apoptotic bodies. The type and amount of cell death influences the recruitment of distinct reactive cells releasing idiosyncratic repertoires of cytokines. These inflammatory cells are differently able to both take up and present the antigen secreted by transfected cells and cross present antigen- expressing cell debris released by the dying cells. The Igea Cliniporator is a fixed voltage, clinical device adopted to improve the penetration of bleomicin into cancer lesions.21 It can also be set to improve DNA vaccination. A morphological analysis of the events following intramuscular injection and electroporation of a plasmid22 shows that damaged myofibers first become infiltrated by granulocytes, then by macrophages, dendritic cells (DC) and later by B and T cells (Fig. 1). The numerous contacts between macrophages, DC, T cells and antigen expressing myofibers suggests that both a direct antigen presentation and macrophage and DC-mediated antigen cross- presentation may occur in the electroporated muscle. By contrast, the reaction of the draining lymph nodes is weak. Exogenous Adjuvants As the environment plays a major role in modulating the res- ponse that ensues after delivery of a plasmid, we have set out to determine whether adjuvants enhance the immune response REVIEW elicited in mice by the intramuscular injection or electropora- tion of a plasmid (RRTErbB-2) coding for the extracellular and transmembrane domains of rat ErbB-2 oncogene.11,23,24 LAG-3 (lymphocyte activation gene-3/CD223) is a type I transmembrane protein associated with the T cell receptor (TCR)–CD3 complex. Administration of soluble LAG-3, gener- ated by fusing the extracellular domain of murine LAG-3 to murine IgG2a Fc portion, in the area in which RRTErbB-2 has been injected potentiates the vaccination by eliciting a stronger CD8+ T cell activity, a sustained secretion of both interferon-gamma (IFNc) and IL-4, and the expansion of CD8+/CD11b+/CD28+ effector and CD8+/CD11b+/CD28- effector-memory T cells. Combination of plasmid injection with soluble LAG-3 also leads to higher titers of anti-rat ErbB-2 antibodies with a faster shift toward IgG2a. In mammary cancer prone rat ErbB-2-transgenic BALB/c mice (BALB-neuT mice) this enhanced response goes along with a sustained protection against the onset of mammary lesions.25 Remarkably, even the higher immune response that follows RRTErbB-2 electroporation in BALB-neuT mice is further enhanced by intravenous administration of BAT monoclonal antibodies (mAb) (CureTech). The stronger anti-rat ErbB-2 T cell and antibody-mediated immune response thus elicited results in even better protection.26 These BAT mAb bind the programmed death-1 (PD-1) receptor of the B7 family expressed on T cells after activation.27 Following the interaction with its ligands (PDL-1 and PDL-2) PD-1 downregulates TCR signals and promotes T cell anergy and apoptosis. By blocking this suppressive loop, BAT mAb enhance T-cell response and leads to potent anti-tumor immunity both in mouse models and in patients with advanced hematologic cancer.27 Lactoferrin, a glycoprotein of the transferrin superfamily, plays several distinct activities in immunity. We have shown that repeated oral administrations of a recombinant human lactoferrin (Talactoferrin, TLF, Agennix) hamper the onset of mammary carcinomas in BALB-neuT mice.28 Association of oral TLF with RRTErbB-2 electroporation extends tumor-free interval, decreases the number of palpable tumors/mouse, and augments vaccine-induced response as compared with sole RRTErbB-2 electroporation. Build-In Adjuvants Hypomethylated CpG dinucleotide-containing motifs present or inserted in the bacterial plasmid trigger innate immune activities and help to shape the adaptive response elicited by the vaccine. Similarly, pattern recognition receptors that sense intracellular DNA can be used to harness the intrinsic immune- stimulating properties of DNA vaccines. A plasmid encoding the cytosolic DNA-dependent activator of interferon regulatory factors co-injected with antigen-encoding plasmids enhances antigen-specific proliferation and induction of effector and memory T cells.29 In principle, cytokines are excellent adjuvants for DNA vaccines, either as recombinant proteins or as embodied genes. They may increase the response and thus allow selective activation www.landesbioscience.com OncoImmunology 317