Abstract
BackgroundExperimental models using DNA vaccine has shown that this vaccine is efficient in generating humoral and cellular immune responses to a wide variety of DNA-derived antigens. Despite the progress in DNA vaccine development, the intracellular transport and fate of naked plasmid DNA in eukaryotic cells is poorly understood, and need to be clarified in order to facilitate the development of novel vectors and vaccine strategies.Methodology and Principal FindingsUsing confocal microscopy, we have demonstrated for the first time that after plasmid DNA uptake an inhibition of the acidification of the lysosomal compartment occurs. This lack of acidification impaired antigen presentation to CD4 T cells, but did not alter the recruitment of MyD88. The recruitment of Rab 5 and Lamp I were also altered since we were not able to co-localize plasmid DNA with Rab 5 and Lamp I in early endosomes and late endosomes/lysosomes, respectively. Furthermore, we observed that the DNA capture process in macrophages was by clathrin-mediated endocytosis. In addition, we observed that plasmid DNA remains in vesicles until it is in a juxtanuclear location, suggesting that the plasmid does not escape into the cytoplasmic compartment.Conclusions and SignificanceTaken together our data suggests a novel mechanism involved in the intracellular trafficking of plasmid DNA, and opens new possibilities for the use of lower doses of plasmid DNA to regulate the immune response.
Highlights
The use of plasmid DNA in vaccinology and gene therapy has shown that plasmid DNA is efficient in generating humoral and cell-mediated immune responses to a wide variety of DNA-derived antigens in various animal models [1]
The results showed that in J774 cells, after 15 minutes, the plasmid DNA and the transferrin were colocalized in early endosomes in peripheral cytoplasmic compartments (Figure 1C)
At all time points (1 h–120 h) DNA was localized in vesicles and after 96 h the vesicles containing plasmid DNA were localized around nuclei (Figure 3F and 3I). These results demonstrate that after endocytosis plasmid DNA remained in the vesicles until it arrived adjacent to the nucleus, suggesting that bulk of the DNA does not escape from the endo-lysososomes to the cytoplasm (Figures 3 and 4)
Summary
The use of plasmid DNA in vaccinology and gene therapy has shown that plasmid DNA is efficient in generating humoral and cell-mediated immune responses to a wide variety of DNA-derived antigens in various animal models [1]. Investigations of the intracellular route of plasmid DNA adsorbed on cationic polymer, cationic lipid or encapsulated in polymers have been widely explored [8,9,10] These studies suggested that formulation with positive charge such as lipoplex and polyplex were internalized by clathrin-dependent endocytosis [11]. The internalization of DNA entrapped in PLGA polymer occurs by phagocytosis (for microparticles, 1–10 mm) or through fluid phase pinocytosis and clathrin-coated pits (for nanoparticles, 70 nm) [13,14,15] In these models, after its uptake, it is thought that plasmid DNA escapes to the cytoplasm, and that this process occurs through the disruption of the endo-lysosomal membrane. Despite the progress in DNA vaccine development, the intracellular transport and fate of naked plasmid DNA in eukaryotic cells is poorly understood, and need to be clarified in order to facilitate the development of novel vectors and vaccine strategies. Taken together our data suggests a novel mechanism involved in the intracellular trafficking of plasmid DNA, and opens new possibilities for the use of lower doses of plasmid DNA to regulate the immune response
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