Plasmid-based DNA Vaccination Protocol

To generate plasmids for DNA vaccination, a previously described plasmid expression vector designated as pT.neo was used [26] (link). The antigenic peptide sequences were inserted at the 3′ EcoRI/XbaI flanking site of the T helper epitope of pT.neo. For co-immunization studies, a previously described plasmid encoding for murine interferon-γ (pmIFN-γ) was used [27] (link). The plasmids were produced by transforming DH5a Escherichia coli cells and purified, after sequence analysis, using EndoFree Plasmid Maxi or Giga kits (Qiagen). Each lot of plasmid DNA had a A₂₆₀/A₂₈₀ ratio ≥1.8, as determined by UV spectrophotometry, endotoxin content ≤0.1 EU/µg DNA, as determined by Limulus Amebocyte Lysate test (Associates of Cape Cod) and a predominantly supercoiled form.
For comparison, two selected peptides were conjugated to keyhole limpet haemocyanin (KLH) to increase their antigenicity and immunogenicity.