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DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA. [ABSTRACT FROM AUTHOR]

Abstract: A plasmid rendered replicative in mammalian cells by inserting the Porcine circovirus 2 (PCV2) origin of replication and replicase gene (Ori-rep) has been previously constructed. The aim of the present study was to evaluate if the replication capacity of this plasmid could be advantageously used to improve the protective immunity induced by DNA vaccination. In this case we used the porcine Pseudorabies virus (PrV) DNA vaccination model. The replicative capacity of the DNA vaccine did not improve the protective immunity against PrV in pigs, but on the contrary the presence of the PCV2 Ori-rep sequence was harmful in the induction of this immunity compared to an equivalent but non-replicative DNA vaccine. In addition, the distribution and the persistence of the replicative and non-replicative plasmids inside the body were the same. This is the first study showing an in vivo deleterious effect of the replicative active PCV2 Ori-rep on the natural and specific protection against PrV infection. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 (PCV-2) is involved in several diseases named porcine circovirus-associated diseases and is transmitted by oro-faecal route. In this study we inoculated porcine-circovirus free piglets by mucosal routes (intratracheal or oro-nasal routes) with a plasmid carrying two copies of PCV-2 genomic DNA and compared the results to the intramuscular route. We observed that this PCV-2 naked DNA serves as template for viral replication and infectious PCV-2 particles are detected in the whole body after parenteral (intramuscular) or mucosal (intratracheal or oro-nasal) delivery. These results suggest that PCV-2 genome could play a role in in vivo transmission. [Copyright &y& Elsevier]

Abstract: DNA vaccination has been widely studied to develop new, alternative, efficient and safe vaccines for humans and animals. Many efforts have been made to increase the immunising potential of these vaccines and three veterinary vaccines are now available on the market. Much work is also being dedicated to develop effective DNA vaccines for humans. However, this new vaccination technique raises issues concerning biosafety due to the nature of the vector, i.e. a DNA molecule that contains sequences of prokaryotic origin (e.g. genes for antibiotic resistance). This review describes the development of the new generation of DNA vectors that are partially or completely devoid of elements of prokaryotic origin and outlines the results of studies on the fate of plasmids after their injection in vivo. [Copyright &y& Elsevier]

Abstract: In this study, a method was developed to measure replication rates of rolling-circle replicon-based plasmids in eukaryotic cells. This method is based on the discriminative quantitation of MboI-resistant, non-replicated input plasmids and DpnI-resistant, replicated plasmids. To do so, porcine circovirus type 2 (PCV2) replicon-based plasmids were constructed. These plasmids contained the PCV2 origin of replication, the PCV2 Rep promoter and the PCV2 Rep gene. The results show that the replication rate depends on the length of the PCV2 replicon-based plasmid and not on the respective position of the Rep promoter and the promoter of the gene of interest that encodes the enhanced green fluorescent protein (eGFP). In all cases, it was necessary to add the Rep gene encoded by a plasmid and cotransfected as a replication booster. This method can evaluate the replication potential of replicon-based plasmids quickly and is thereby a promising tool for the development of plasmids for vaccine purposes. [Copyright &y& Elsevier]