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European Biopharmaceutical Review

Prevention is Better than Cure

 Prevention is Better than Cure

Vaccination is historically one of the most important methods for the prevention of infectious diseases in humans and animals. Despite the encouraging results obtained with conventional vaccines, based on whole killed or live attenuated micro-organisms and purified components of pathogens, the recent studies about the immune system biology have led to new strategies

In the last decade, naked DNA vaccination is emerging as a promising approach for the introduction of foreign antigens into the host, inducing an immune response not only against infectious diseases, but also against malignant tumours. This article will give a brief review of the DNA vaccination strategy mediated by electrotransfer (ET), reporting mechanism of action and the recent literature supporting this strategy as an effective method to improve DNAbased vaccination protocols against infectious and cancer diseases.

DNA Vaccines and Electrotransfer

DNA plasmid vaccines offer several advantages when compared to traditional vaccines. DNA is a fl exible molecule that can be manipulated in several ways by genetic engineering for increasing antigen expression, immunogenicity and uptake by recipient cells. The antigen can be chemically synthesised and cloned directly into the plasmid vaccine, simplifying the operations of amplifi cation by molecular techniques and avoiding working with potentially dangerous live antigen source. Besides the easy manipulation, DNA vaccines are very stable at room temperature, and do not require any particular storage conditions, which makes them ideal candidates for long-term delivery worldwide.

DNA vaccines are ideally delivered into the skeletal muscle for easy accessibility and the good vasculature of this organ (1). In mammals, the skeletal muscle represents approximately 30 per cent of the body mass, and muscle fi bres are good targets for DNA transfection. Muscle tissue is made by stable and large syncytial cells containing several nuclei that can actively take part in immune reactions. Immunisation with DNA induces an immune response with a simple mechanism. Once the DNA vaccine is delivered into the skeletal muscle, the plasmid DNA is taken up by the resident dendritic cells (DCs) and by the muscle fi bres. While transfected muscle cells act as a permanent antigen reservoir, as well as a target of immune effector cells, resident DCs will leave the muscle tissue and move to the closest draining lymph nodes in order to process and present the antigen to T lymphocytes. DCs are specialised in capturing extracellular antigens by endocytosis and pinocytosis mechanisms and, following antigen uptake, they undergo a complex multi-step maturation process. DC maturation also depends on microbial and pathogensderived signals, which increase their capacity to migrate towards the draining lymph nodes. While DCs move to the lymphoid organs, they interact with various chemokines which contribute further to their maturation process.

Once in the lymph nodes, DCs shift from an antigen-capturing cell to a T cell-sensitising cell, being capable of presenting antigen in association with class 1 and class 2 major histocompatibility complex (MHC) molecules to cytotoxic T cell (CTLs) and T-h lymphocytes. Interaction between

the DC and the T lymphocyte induces formation of the immunological synapse via complex MHC-antigen-T cell receptor 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 the form of apoptotic bodies activating the cross-presentation pathway. This route allows presentation of extracellular or exogenous antigens through the MHC-I restriction pathways.

Therefore, extracellular antigens, which normally induce a humoural immune response, can also access the MHC-I compartment through endoplasmic reticulum, leading to simultaneous stimulation of the CTL immune response. Antigen synthesised by DC or skeletal muscle cells can also be released in the extracellular environment and directly activate the B lymphocytes through antigen-antibody interaction (2).

Genetic immunisation confers the same broad immunological advantages as immunisation with live, attenuated vaccines does, without the disadvantages which often occur with live infection, such as reversion to the virulent form and/or incomplete inactivation of live vaccines.

 Nevertheless, plasmid DNA vaccines need to be improved due to their poor immunogenicity when administered as unformulated intramuscular injections. Different strategies can be used for enhancing the plasmid DNA vaccine potency.

Exposure of biological cells to an external electric field results in an increased permeability of cell membranes, referred to as ‘electroporation’. This is a technology platform widely used in different scientific domains such as medicine, biotechnology and environmental preservation (3). Studies on this issue are promoted at European level, through cooperation in science and technology (COST) action aims.

Current data suggest that the DNA vaccine efficacy can be increased significantly by electrotransfer (4). The transient increase in the permeability of cell membranes, when exposed to electric field pulses, improves the gene delivery and the stimulation of both humoural and cellular immunity, enhancing their mechanisms.

Interest in the application of ET to DNA vaccination protocols has grown rapidly over the last few years for several reasons. It has been demonstrated that ET allows an improved uptake of DNA in tissue cells, especially if used in combination with hyaluronidase (5). This enzyme – able to degrade hyaluronan, an ubiquitous glycosaminoglycan of the extracellular matrix surrounding muscular fibres and skin–leads to a better penetration and spread of DNA in the skeletal muscle (6,7). In clinical practice, it is used to increase the absorption and dispersion of other injected drugs in human patients, adopted in the nonpharmacological treatment of extravasation of selected antineoplastic agents and in extravasated contrast media management (8,9).

Although the mechanisms of electrogene transfer have not been completely clarified it is supposed that a higher

DNA uptake in vivo is possible thanks to the enhancement of cell membrane permeabilisation and electrophoretic movement of DNA molecules into the target cells (10). Moreover, if ET is applied in muscle cells, these work as a platform for antigen production within the skeletal muscle (11). A combination of both these events facilitates target cell transfection, with the result of a higher synthesis of the gene of interest and an intensification of the immune response to the encoded protein.

In respect to a simple administration of DNA vaccines through intramuscular injection, ET is responsible for a significant increase in antibody titre antigen-specific T cell frequency and induction of several T cell effector functions (12-14).

Adjuvancy is a crucial step to take into consideration in vaccination protocols (15). A study performed on DNA vaccination mediated by ET demonstrated that the concomitant injection of plasmid DNA and ET is crucial for the adjuvant effect exerted by ET, which is responsible for eliciting antigenspecific IgG2a antibody production and Th-1 biased immune responses (16). Recently it has also been reported that ET is a crucial event through which – as it induces transient morphological changes and a local moderate damage in the treated muscle – it is possible to generate an early production of endogenous cytokines responsible for signalling danger at the local level. The activation of a danger pro-inflammatory pathway and the recruitment of inflammatory cells result in T lymphocyte migration, indicating ET is able to recruit and trigger cells involved in antigen presentation, especially if used in combination with hyaluronidase (17,18).

Following the above considerations, ET is recognised as being helpful in DNA vaccination protocols for increasing the potency and safety of this therapeutic approach.

Conclusion

Naked DNA vaccination is emerging as a promising approach for its application in human patients. The problem related to their low efficacy is gradually being overcome by ET, and many clinical trials are under investigation. Phase 1 and some Phase 2 clinical trials based on DNA vaccination mediated by ET against infectious diseases and malignant tumours are currently on-going to test their safety and efficacy, in order to make this strategy useful and practicable in the future (19).

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Emanuela Signori has an MSc in Natural Science, post graduate training in Molecular Biology and Molecular Medicine, and a PhD in Experimental Oncology and Molecular Pathology. She is the Laboratory Head at CNR-IFT, Laboratory of Molecular Pathology and Experimental Oncology, and acting Professor of General Pathology at University Campus Bio-Medico (UCBM) of Rome, School of Medicine. Emanuela has also authored over 30 peer-reviewed papers and four book chapters. Her research activity is focused on translational molecular medicine for novel targeted therapies, to pass from bench to bedside by identifying therapeutic molecules and innovative strategies for their administration in preclinical protocols.

Email: emanuela.signori@ift.cnr.it

Vito Michele Fazio, MD, is a specialist in oncology and Full Professor of Laboratory Medicine, Chairman of General Pathology, Director of Laboratory of Molecular Medicine and Biotechnology, and Director of the Residency in Clinical Pathology at the University Campus Bio-Medico (UCBM) of Rome. He is also Deputy Scientific Director and Director of the Laboratory of Oncology at the Scientific Institute and Hospital IRCCS H 'Casa Sollievo della Sofferenza', San Giovanni Rotondo, Italy. Vito is a Professor of Applied Immunology in the specialist degree course in Medical Biotechnology and Molecular Medicine, University of Bari. He has authored over 110 scientifi c publications, patents and books.

Email: fazio@unicampus.it
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