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Mucosa are the routes of entry of most pathogens into animals' organisms. Reducing the important global burden of mucosal infectious diseases in livestock animals is required in the field of veterinary public health. For veterinary respiratory pathogens, one possible strategy is the development of intranasal (IN) DNA vaccination. The aim of this study was to assess the feasibility of IN DNA vaccination in pigs, an important species in livestock production industry, and a source of zoonotic diseases. To achieve this goal, we used a DNA vaccine against pseudorabies virus (PrV) encoding the immunogenic glycoprotein B (pcDNA3-gB plasmid). When pigs were inoculated with the naked DNA vaccine through the IN route, PrV-specific IgG and IgA type antibodies were detected in porcine sera. Interestingly, mucosal salivary IgA antibodies against PrV were also detected, at similar levels to those measured following intramuscular injection (positive controls). Furthermore, the IN delivery of pcDNA3-gB combined with PLGA-PEI nanoparticles resulted in similar levels of antibodies but was associated with an increase in the duration of detection of mucosal IgA for 2 out of 3 pigs. Our results suggest that there is room to improve the efficacy of IN DNA vaccination in pigs through optimization of IN inoculations, for example by using nanoparticles such as PLGA-PEI. Further studies will be dedicated to optimizing and testing the protective potential of IN DNA vaccination procedures against PrV., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

DNA vaccination is a promising alternative strategy for developing new human and animal vaccines. The massive efforts made these past 25 years to increase the immunizing potential of this kind of vaccine are still ongoing. A relatively small number of studies concerning poultry have been published. Even though there is a need for new poultry vaccines, five parameters must nevertheless be taken into account for their development: the vaccine has to be very effective, safe, inexpensive, suitable for mass vaccination and able to induce immune responses in the presence of maternal antibodies (when appropriate). DNA vaccination should meet these requirements. This review describes studies in this field performed exclusively on birds (chickens, ducks and turkeys). No evaluations of avian DNA vaccine efficacy performed on mice as preliminary tests have been taken into consideration. The review first describes the state of the art for DNA vaccination in poultry: pathogens targeted, plasmids used and different routes of vaccine administration. Second, it presents strategies designed to improve DNA vaccine efficacy: influence of the route of administration, plasmid dose and age of birds on their first inoculation; increasing plasmid uptake by host cells; addition of immunomodulators; optimization of plasmid backbones and codon usage; association of vaccine antigens and finally, heterologous prime-boost regimens. The final part will indicate additional properties of DNA vaccines in poultry: fate of the plasmids upon inoculation, immunological considerations and the use of DNA vaccines for purposes other than preventing infectious diseases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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 © 2012 Elsevier B.V. All rights reserved.)

This study was performed to determine whether electroporation can be used to enhance the efficacy of a DNA vaccine against pseudorabies virus (PrV) in pigs. Immune responses to PrV were measured in pigs following a single intramuscular injection of plasmids encoding PrV glycoprotein B, with or without electroporation. Plasmid injection coupled with electroporation increased production of specific antibodies against PrV and peripheral blood mononuclear cells proliferated in response to stimulation with PrV glycoproteins. These results show that electroporation can improve the performance of a DNA vaccine against PrV in pigs. However, additional work is required to maximise the effectiveness of the vaccination protocol., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 2010 Elsevier Ltd. All rights reserved.)

DNA vaccination against Foot-and-Mouth Disease Virus (FMDV) is an attractive and alternative strategy to the use of classical inactivated viral vaccines. The injection of a pcDNA3.1-based DNA vaccine encoding for FMDV P1-2A3C3D and GM-CSF proteins had previously been shown to induce the production of neutralizing antibodies against FMDV and partially protect swine against an experimental challenge. Based on the induction of FMDV humoral immune responses, the aim of the present study was to see if the Sindbis virus derived plasmid (pSINCP) backbone could advantageously replace pcDNA3.1 in DNA immunization against FMDV in swine. For this purpose, groups of 3 or 4 pigs received three injections by intramuscular route, intradermal route or an association of both routes, at 2-3 week intervals. The pcDNA3.1-based DNA vaccine was shown to induce the production of higher amounts of FMDV-neutralizing antibodies after intradermal injection. Intramuscular injection of the same vaccine, or intramuscular (IM) and/or intradermal (ID) injection of the pSINCP-based DNA vaccine resulted in a significantly lower induction of FMDV-neutralizing antibodies. In conclusion, the humoral immune response of a DNA vaccine encoding for FMDV P1-2A3C3D was not improved by the pSINCP backbone and was higher when the plasmids were injected by the intradermal route.

To evaluate the feasibility of using pseudorabies virus (PrV) glycoprotein B (gB) as a carrier of foot and mouth disease virus (FMDV) antigens in DNA immunization, FMDV B- and T-cell epitopes were inserted either between the two B-cell epitopes of the N-term subunit of PrV-gB (BT-PrV-gB-N-term construct) or within the B-cell epitope of the C-term subunit of PrV-gB (BT-PrV-gB-C-term construct). Two animal experiments were performed, each with three injections of plasmids 2 weeks apart, followed by a booster inoculation of peptides corresponding to the FMDV epitopes. Control groups of pigs were injected with plasmids encoding either PrV-gB or FMDV-BT, or with empty-pcDNA3. The results of both assays were combined. Significant titers of FMDV neutralizing antibodies were detected after the peptides boost in groups injected with the BT-PrV-gB-C-term construct. Insignificant amounts were detected in groups injected with the BT-PrV-gB-N-term and FMDV-BT constructs. PBMCs from the BT-PrV-gB-N-term groups, isolated after the peptide boost injection, produced IFN-gamma and IL-4 mRNAs in vitro when stimulated with FMDV peptides. This was not observed with the other groups. These results imply that PrV-gB can be used to carry FMDV antigens in a DNA vaccine.

Previous biodistribution studies of plasmids following intramuscular or intradermal injections of DNA vaccines have been performed in mice, rats or rabbits, but not in large mammals. The aim of the present study was to determine the biodistribution of plasmids in swine using the PRV-specific DNA vaccination model consisting of a single intramuscular (i.m.) injection of three plasmids individually encoding glycoproteins gB, gC and gD. The weak bioavailability of the plasmids (less than 10%) after i.m. injection was consistent with the tissue distribution study. Plasmids remained in the injected muscle for at least 4 weeks and were also detected in liver, spleen, kidney, lung, remote muscle, lymph nodes and ovaries for shorter periods. Differences in persistence, apparent elimination half-lives and clearance in blood were observed between the three plasmids. In conclusion, the three plasmids behaved differently and were transiently detected in most of the organs tested. The exact persistence in the injected muscle was not determined but exceeded 4 weeks. To date this is the first published DNA vaccine tissue distribution study in large animals.

No pseudorabies virus (PRV)-specific neutralizing or immunoglobulin G1-type antibodies were detected in sera 5 days after injection of a DNA vaccine against PRV infection in pigs. PRV-stimulated peripheral blood mononuclear cells produced gamma interferon mRNA in vitro. Two out of five pigs recovered from lethal PRV infection without attenuation of nasal viral excretion.

Plasmids encoding ubiquitinated (ubi-) or non-ubiquitinated (non-ubi-) glycoproteins of Pseudorabies virus (PRV) were used for vaccination of pigs. We found that the fusion of ubiquitin to viral glycoproteins increased their degradation in proteasomes in vitro, in which ubiquitin plays a key role. In the animals immunized with the plasmids encoding PRV ubi-glycoproteins and then challenged with PRV, we detected a slightly decreased cellular immune response on days 13 and 19 after immunization and a reduced nasal excretion of infectious virus on day 2 after the challenge. Afterwards, no effect of the ubiquitination of the glycoproteins on humoral or cellular immunity and on excretion of infectious virus was observed. Similarly, no effect of the ubiquitination on protective abilities of PRV glycoproteins was found.

The present study demonstrates the protective potential of a novel prime-boost vaccination strategy of pigs against lethal Pseudorabies virus (PRV; Herpes suid 1) infection. Animals were primed with Sindbis virus-derived plasmids that express viral glycoproteins gC and gD (gC- and gD-pSIN) and subsequently booster immunized with Orf virus (ORFV; Parapoxvirus) recombinants expressing gC and gD (D1701-VrVgC and -VrVgD). The prime-boost vaccination induced strong humoral and cellular-like PRV-specific immune responses. All prime-boost vaccinated pigs survived the lethal challenge infection without PRV-specific clinical symptoms and presented excellent body weight loss attenuation. Most notably, nasal shedding of challenge virus was reduced by more than about 3log(10), clearly reducing the risk of infection of non-immunized pigs.

New technology, and in particular new molecular biology tools, have opened up access to new generations of vaccines or control tests. DNA vaccines are of particular interest since only the gene is injected and, as a consequence, specific risks are associated with this kind of vaccination. Some strategies are presented here to improve DNA vaccine efficacy, which can in some cases result in the reduction of the quantity of plasmid needed and therefore limits the risk. Specific tools to study the distribution of plasmids inside the body as well as tools to detect plasmid insertion inside the host genome have been developed. A final part of this paper will present briefly the new tools which have been or can be developed to detect contaminants in vaccines.

This study evaluated the adjuvant activity of a porcine-specific type A CpG-containing oligodeoxynucleotide (CpG-ODN) in a DNA-vaccine against Pseudorabies virus (PrV) infection. This vaccine, composed of three independent plasmids encoding PrV gB, gC and gD glycoproteins, was injected alone or was co-injected with CpG-ODN or control GpC-ODN. CpG-ODN improved the PrV-specific humoral immune response and provided better clinical protection against lethal PrV-infection. The PrV-specific cell-mediated immune response seemed also to be increased.

This study compared protection of pigs against lethal Pseudorabies virus (PrV) infection induced by a single injection of various quantities of Sindbis virus-derived plasmids encoding PrV glycoproteins gB, gC and gD. Pigs were injected with 340, 68 or 13 microg of each plasmid. Few immune differences were observed after DNA injection and more importantly the pigs of the three groups were equally protected against virulent PrV infection. Single-shot injection of 13 microg of each PrV glycoprotein encoding Sindbis virus-derived plasmid is able to effectively protect pigs from PrV infection.