Daniel Dory

Biosafety of DNA vaccines: New generation of DNA vectors and current knowledge on the fate of plasmids after injection

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.

Current strategies for subunit and genetic viral veterinary vaccine development

Developing vaccines for livestock provides researchers with the opportunity to perform efficacy testing in the natural hosts. This enables the evaluation of different strategies, including definition of effective antigens or antigen combinations, and improvement in delivery systems for target antigens so that protective immune responses can be modulated or potentiated. An impressive amount of knowledge has been generated in recent years on vaccine strategies and consequently a wide variety of antigen delivery systems is now available for vaccine research. This paper reviews several antigen production and delivery strategies other than those based on the use of live viral vectors. Genetic and protein subunit vaccines as well as alternative production systems are considered in this review.

Control strategies against Campylobacter at the poultry production level: biosecurity measures, feed additives and vaccination.

Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union, and ranks second in the United States only behind salmonellosis. In Europe, there are about nine million cases of campylobacteriosis every year, making the disease a major public health issue. Human cases are mainly caused by the zoonotic pathogen Campylobacter jejuni. The main source of contamination is handling or consumption of poultry meat. Poultry constitutes the main reservoir of Campylobacter, substantial quantities of which are found in the intestines following rapid, intense colonization. Reducing Campylobacter levels in the poultry chain would decrease the incidence of human campylobacteriosis. As primary production is a crucial step in Campylobacter poultry contamination, controlling the infection at this level could impact the following links along the food chain (slaughter, retail and consumption). This review describes the control strategies implemented during the past few decades in primary poultry production, including the most recent studies. In fact, the implementation of biosecurity and hygiene measures is described, as well as the immune strategy with passive immunization and vaccination trials and the nutritional strategy with the administration of organic and fatty acids, essential oil and plant‐derived compound, probiotics, bacteriocins and bacteriophages.

Identification of Novel Vaccine Candidates against Campylobacter through Reverse Vaccinology

Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union. Human cases are mainly due to Campylobacter jejuni or Campylobacter coli, and contamination is associated with the handling and/or consumption of poultry meat. In fact, poultry constitutes the bacterias main reservoir. A promising way of decreasing the incidence of campylobacteriosis in humans would be to decrease avian colonization. Poultry vaccination is of potential for this purpose. However, despite many studies, there is currently no vaccine available on the market to reduce the intestinal Campylobacter load in chickens. It is essential to identify and characterize new vaccine antigens. This study applied the reverse vaccinology approach to detect new vaccine candidates. The main criteria used to select immune proteins were localization, antigenicity, and number of B-epitopes. Fourteen proteins were identified as potential vaccine antigens. In vitro and in vivo experiments now need to be performed to validate the immune and protective power of these newly identified antigens.

DNA vaccination of poultry: The current status in 2015.

Abstract 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.

Foot-and-Mouth Disease Virus neutralizing antibodies production induced by pcDNA3 and Sindbis virus based plasmid encoding FMDV P1-2A3C3D in swine

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.

Effect of granulocyte-macrophage colony-stimulating factor on post-weaning multisystemic wasting syndrome in porcine circovirus type-2-transfected piglets.

Post‐weaning multisystemic wasting syndrome (PMWS) is a complex disease syndrome in swine, affecting nursery and fattening pigs. Although ongoing evidence suggests that porcine circovirus type‐2 (PCV2) is the causal agent of PMWS, the host immune system appears to have a crucial role in the PMWS pathogenesis of PCV2‐affected pigs. Owing to difficulties in producing a biologically pure form of PCV2 devoid of the other viral agents commonly present in swine tissues, we decided to use a tandem‐cloned PCV2 DNA providing highly pure grade reagent in order to monitor the virulence of PCV2 alone or with an immunostimulating co‐factor, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). A single intramuscular injection of tandem‐cloned PCV2 DNA into 5‐week‐old piglets produced plasmid to viral genome progeny and infectious particles as early as 8 days post‐injection in all the organs tested (the lung, the tonsil and the inguinal, mesenteric, bronchial and upper‐right axial lymph nodes). The initial plasmid load was not detected with the help of primers designed to specifically detect the acceptor plasmid, thus confirming the replication of the viral genome. Despite the presence of a high level of PCV2 genome copies in the lymphoid organs – the tonsil and the lung – and the presence of infectious particles, no detectable clinical manifestations or pathological lesions were observed in the transfected pigs over the period of observation, regardless of whether they had been co‐injected with plasmid containing GM‐CSF DNA or had received plasmid containing PCV2 DNA alone. GM‐CSF encoding DNA injection had no significant effect on viral replication or on the production of viral particles and appearance of the disease.

Replication efficiency of rolling-circle replicon-based plasmids derived from porcine circovirus 2 in eukaryotic cells

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.

Pseudorabies virus glycoprotein B can be used to carry foot and mouth disease antigens in DNA vaccination of pigs.

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.

Promising new vaccine candidates against Campylobacter in broilers

Campylobacter is the leading cause of human bacterial gastroenteritis in the European Union. Birds represent the main reservoir of the bacteria, and human campylobacteriosis mainly occurs after consuming and/or handling poultry meat. Reducing avian intestinal Campylobacter loads should impact the incidence of human diseases. At the primary production level, several measures have been identified to reach this goal, including vaccination of poultry. Despite many studies, however, no efficient vaccine is currently available. We have recently identified new vaccine candidates using the reverse vaccinology strategy. This study assessed the in vivo immune and protective potential of six newly-identified vaccine antigens. Among the candidates tested on Ross broiler chickens, four (YP_001000437.1, YP_001000562.1, YP_999817.1, and YP_999838.1) significantly reduced cecal Campylobacter loads by between 2 and 4.2 log10 CFU/g, with the concomitant development of a specific humoral immune response. In a second trial, cecal load reductions results were not statistically confirmed despite the induction of a strong immune response. These vaccine candidates need to be further investigated since they present promising features.