**1.3 Ex-vivo loaded dendritic cells**

Antigen Presenting Cells-based vaccines represent another explored field in vaccine research. With this approach, DC are harvested from the patient, pulsed with antigens or transfected with genes encoding these antigens, and readministered to the patient. This vaccine strategy has the potential to augment presentation through the MHC-class I pathway and subsequently drive the expansion of tumour-specific CTLs. In translational studies with melanoma patients, DC vaccines have demonstrated a keen ability to elicit detectable immune responses. However, such responses often fail to elicit substantial clinical responses. As it is often difficult to discern the relative contributions of DCs and effector T cells in these situations, a thorough investigation of the *in vivo* interactions between these immune cell populations may be required before a complete understanding of DC role (Palucka et al. 2007).

#### **1.4 Genetic vaccination**

Recently, new methods of vaccination such as those based on gene transfer have emerged. Genetic vaccination originates from gene therapy. The objective of genetic vaccination is to transfer in the host a gene encoding for the disease target antigen with the aim to induce a specific immune response, whereas the goal of gene therapy is to ensure production of a protein which is lacking or defective in the host. To date, the vast majority of gene therapy clinical trials have addressed cancer (66.5%), cardiovascular diseases (9.1%) and infectious diseases (6.5%). For infectious diseases, a total of 85 gene therapy trials have been carried out, the majority of these trials being performed on human immunodeficiency virus infection, tetanus, cytomegalovirus and adenovirus infections (Chiarella et al. 2008a).

Current techniques of gene transfer in mammals include packaging the DNA into carriers for gene delivery. The ideal carriers for gene delivery should be safe and yet ensure that the DNA survives the extra and intracellular environment, efficiently transfer to the appropriate cellular compartments assuring good and long-lasting expression levels.

Presently, viral vectors are more efficient than non-viral systems, achieving high levels of efficiency, estimated around 90%, for both gene delivery and expression. However, immunogenicity, inflammatory reactions, problems associated with scale-up costs and, more important, the risk of integration in the host genome, are limiting their clinical use in preclinical and clinical protocols respect to the past: i.e. during 2000 year, around 75% of clinical protocols involving gene therapy used recombinant virus-based vectors for DNA delivery (Chiarella et al. 2008a). In the last years, lot of clinical trials pointed out that the use of viral vectors as antigen delivery systems has numerous other drawbacks such as toxicity, recombination, precedent host immunity, higher immunogenicity in comparison to the

DNA Vaccination by Electrogene Transfer 173

epitope and antigen choice, selection and use of new adjuvants and different delivery

In table 1 the advantages and disadvantages of DNA vaccines are listed.

Antigen presentation MHC-I, MHC-II, cross-priming Immune Response Humoural and cytotoxic

Stability Stable at various temperature (RT)

Applicability Prophylaxis and therapy of disease

Table 1. Advantages and disadvantages of plasmid DNA vaccines.

**2.2 Mechanism of action and induction of the immune response** 

Manufacture Easy and fast

helper (Th), cytotoxic (CTL) and B lymphocytes.

Immunogenicity Weak

**Characteristic Advantage/disadvantage of plasmid DNA vaccines**  Antigen In vivo antigen synthesis with native conformation

Risk Does not induce the disease related to the encoded antigen

The crucial event responsible for the initiation of an immune response against a foreign antigen is recognition by specialized cells namely the antigen presenting cells (APCs), uptake and presentation of the antigen to naïve lymphocytes and induction of effector T

In this context the mechanism of action of DNA vaccines looks very simple. Once the DNA vaccine is delivered into the skeletal muscle, the plasmid DNA is taken up by the resident DCs and by the muscle fibres. While transfected muscle cells behave as permanent antigen reservoir as well as target of immune effector cells (Payette et al. 2001), resident DCs have the property to leave the muscle tissue and move to the closest draining lymph nodes in order to process and present the antigen to T lymphocytes (**Fig. 1**). DCs are specialized in capturing extracellular antigens by receptor-mediated endocytosis and pinocytosis mechanisms and following antigen uptake they undergo a complex multi-step maturation process. DC maturation depends also on the microbial and pathogens-derived signals which increase their capacity to migrate towards the draining lymph node. While DCs move to the lymphoid organs, they interact with various chemokines which contribute further to their maturation process (Palucka et al. 2010). Once in the lymph nodes, DCs shift from an antigen-capturing cell to a T sensitizing cell, being capable to present antigen in association with the class I and class II MHC molecules to CTLs and Th lymphocytes. Interaction between the DC and the T lymphocyte induces formation of the immunological synapse (IS) *via* complex MHCantigen- T cell receptor (TCR) resulting in the clonal expansion of the T lymphocyte and differentiation in T memory cell. Professional DCs can also capture antigens released in the interstitial space by skeletal muscle fibres or in form of apoptotic bodies activating the cross-presentation pathway (**Fig. 1**) (Russo et al. 2000). This route allows presentation of extracellular/exogenous antigens through the MHC-I restriction pathways (Kurts et al. 2010). Therefore, extracellular antigens which normally induce a humoural immune response can also access to the MHC-I compartment through endoplasmic reticulum, leading to simultaneous stimulation of the CTL immune response. Antigen synthesized

Indication of use Infectious disease, allergy, cancer, autoimmune disease

Safety Low risk of recombination and inflammation

methods.

target antigen and limited DNA carrying capacity (Harrington et al. 2002; Ramirez et al. 2000). The recent advances made on the knowledge of the immune system biology have led to consider non-viral systems as naked DNA vaccination an alternative, safer and promising approach for introducing foreign antigens into the host to induce an immune response. At the moment, non-viral systems, especially those based on plasmid DNA delivery, have become increasingly desirable in both basic research laboratories and clinical settings.
