DNA immunization against AnHV-1 has been developed in our laboratory
. To increase the efficacy of the AnHV-1 gC DNA vaccine, DNA-lipid complexes and chitosan-DNA nanoparticles were prepared. In the present study, the fates of these complexes and pcDNA3.1-gC alone following intramuscular injection were investigated in ducklings. To our knowledge, this study is the first assessment of the organ distribution and retention time of plasmid DNA administered in lipid complexes or chitosan nanoparticles in ducklings.
More than three decades ago, Wolff showed that the injection of mouse muscle with a DNA plasmid resulted in significant expression of the protein encoded by the plasmid
. Progress in the field of DNA vaccination has resulted in the development and the marketing of three veterinary DNA vaccines
. The uptake of DNA plasmid by cells upon injection is very inefficient: only a small proportion of the injected material is internalized by cells and results in successful transfection
. Therefore, a number of strategies have been used for increasing DNA vaccine potency. Chemical adjuvants for enhancing plasmid DNA expression include liposomes and cationic polymers, both of which have shown promise for enhancing the expression and immunogenicity of plasmid DNA vaccines in animal models
Our results showed that liposomes universally increased the plasmid DNA copy numbers at the injection site, liver, spleen, heart, brain, bursa of Fabricius, and especially in the enteron (esophagus, duodenum, rectum, and cecum), compared with pcDNA3.1-gC alone. Similarly, chitosan also universally increased the plasmid DNA copy numbers at the injection site, liver, spleen, heart, brain and esophagus. These results indicated that liposomes and chitosan enhance the distribution in tissues. In addition, compared with lipoplex-gC, chitosan-gC mediated production of higher numbers of plasmid DNA copies at the injection site, liver, spleen, heart, brain and esophagus. Nevertheless, compared to lipolex-gC, lower copy numbers were detected in duodenum, rectum, and cecum.
In terms of the effects of time after inoculation, the highest concentrations of DNA were detected all the tissues at 1 h after injection. The number of plasmid copies had decreased by 3–4 orders of magnitude at 1 day after injection. Subsequently, the number of plasmid copies was maintained at a low level and plasmid DNA was still be detected 10 weeks post-injection. Our study is consistent with other studies, which have shown that plasmid remained for several weeks following clearance of the majority of the plasmid within the first 24 h post-inoculation
[26–28]. Additionally, results based on indirect immunohistochemical staining (IHC) have shown that the gC proteins are still found in the liver, bursa of Fabricius, duodenum, caecum and rectum in the intramuscular injection group at 10 weeks post-inoculation
Transfection of cells by chitosan nanoparticles and lipoplexes occurs in three phases: (1) cellular uptake by formation of endosomes into the target cells; (2) release from the endosome; (3) entry of plasmid DNA into the cell nucleus
[30, 31]. Liposomes can protect the nucleic acid from extracellular degradation, ensuring appropriate tissue targeting, and facilitating the delivery of functional DNA into the cell
[32, 33]. As a cationic polymers, chitosan (CS) is an effective, naturally-occurring material used for synthesizing nanoparticles with advantageous properties such as low toxicity, low immunogenicity and excellent biocompatibility
[34, 35]. Accordingly, following inoculation with chitosan/DNA or liposome/DNA complexes, plasmid DNA diffuses from the injection site and/or degrades more slowly because of a liposomes or chitosan depot effect. The liposome/DNA or chitosan/DNA depot is thought to provide some protection against nucleases, thus extending the half-life of the plasmid DNA at the injection site
. It has been shown that formulation of DNA vaccines into liposomes enhances cellular and humoral immunity
[34, 35]. Our previous study also showed that chitosan significantly improved CD4+ and CD8+ T cell responses to at least 6 weeks post-injection in Balb/c mice
. The immune responses to these formulations and protection against Anatid herpesvirus 1 challenge in ducks are currently being analyzed.
Fluorescence-based quantitative real-time PCR (qPCR) is commonly regarded as a straightforward, mature and ubiquitous means of molecular biology
. Probe-based qPCR technology is recommended as the reference technique for monitoring gene transfer biodistribution
. Therefore, in our previous study, we established a qPCR protocol based on the use of a TaqMan™ probe
. In contrast to previous studies, equivalent amounts of DNA (100 μg) were injected in the form of pcDNA3.1-gC, DNA-lipid complexes and chitosan-DNA nanoparticles and 200 ng of gDNA were used in each PCR reaction by preparation of the appropriate dilution ratios. Because we observed that there existed the inhibition due to the gDNA matrix, which affected quantification using qPCR (data not shown). This was consistent with other studies
. In the present study, 100 μg of plasmid was injected for each group. The same amount of each tissue was collected.
In summary, we successfully used chitosan and lipid formulations for DNA vaccination in ducklings. Compared with pcDNA3.1-gC alone, chitosan/DNA and DNA/lipid complexes improved the efficiency of plasmid distribution. The complexes were rapidly absorbed, and extensive and relatively long-term distribution at low concentrations was observed following DNA vaccination in ducklings.