title: Comparative analysis of antigen-targeting sequences used in DNA vaccines.
abstract: Plasmid vectors can be optimized by including specific signals that promote antigen targeting to the major antigen presentation and processing pathways, increasing the immunogenicity and potency of DNA vaccines. A pVAX1-based backbone was used to encode the Green Fluorescence Protein (GFP) reporter gene fused either to ISG (Invariant Surface Glycoprotein) or to TSA (trans-sialidase) Trypanosoma brucei genes. The plasmids were further engineered to carry antigen-targeting sequences, which promote protein transport to the extracellular space (secretion signal), lysosomes (LAMP-1) and to the endoplasmic reticulum (adenovirus e1a). Transfection efficiency was not affected by differences in the size between each construct as no differences in the plasmid copy number per cell were found. This finding also suggests that the addition of both ISG gene and targeting sequences did not add sensitive regions prone to nuclease attack to the plasmid. Cells transfected with pVAX1GFP had a significant higher number of transcripts. This could be a result of lower mRNA stability and/or a lower transcription rate associated with the bigger transcripts. On the other hand, no differences were found between transcript levels of each ISG-GFP plasmids. Therefore, the addition of these targeting sequences does not affect the maturation/stability of the transcripts. Microscopy analysis showed differences in protein localization and fluorescent levels of cells transfected with pVAX1GFP and ISG constructs. Moreover, cells transfected with the lamp and secretory sequences presented a distinct distribution pattern when compared with ISG protein. Protein expression was quantified by flow cytometry. Higher cell fluorescence was observed in cells expressing the cytoplasmic fusion protein (ISG-GFP or TSA-GFP) compared with cells where the protein was transported to the lysosomal pathway. Protein transport to the endoplasmic reticulum does not lead to a decrease in the mean fluorescence values. The secretion signal was only effective when used in conjunction with TSA gene. Therefore, the characteristics of each protein (e.g., presence of transmembrane domains) might influence the efficacy of its cellular transport. This analysis constitutes a useful tool for the optimization of the design of DNA vaccines.
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal.
authors.forename: Joana A
authors.initials: JA
authors.lastname: Carvalho
authors.forename: Adriano R
authors.initials: AR
authors.lastname: Azzoni
authors.forename: Duarte M F
authors.initials: DM
authors.lastname: Prazeres
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Monteiro
mesh_terms: D000941:Antigens / Q000235:genetics*; D019677:Artificial Gene Fusion / Q000379:methods; D001483:Base Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D017422:Sequence Analysis, DNA; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*
chemical_list: D000941:Antigens; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D019444:Vaccines, DNA; D049452:Green Fluorescent Proteins; C069715:trans-sialidase; D009439:Neuraminidase