Evaluation of immunogen delivery by DNA immunization using non-invasive bioluminescence imaging

The efficacy of DNA vaccines is highly dependent on the methods used for their delivery and the choice of delivery sites/targets for gene injection, pointing at the necessity of a strict control over the gene delivery process. Here, we have investigated the effect of the injection site on gene expression and immunogenicity in BALB/c mice, using as a model a weak gene immunogen, DNA encoding firefly luciferase (Luc) delivered by superficial or deep injection with subsequent electroporation (EP). Immunization was assessed by monitoring the in vivo expression of luciferase by 2D- and 3D-bioluminescence imaging (BLI) and by the end-point immunoassays. Anti-Luc antibodies were assessed by ELISA, and T-cell response by IFN-γ and IL-2 FluoroSpot in which mouse splenocytes were stimulated with Luc or a peptide representing its immunodominant CD8+ T-cell epitope GFQSMYTFV. Monitoring of immunization by BLI identified EP parameters supporting the highest Luc gene uptake and expression. Superficial injection of Luc DNA followed by optimal EP led to a low level Luc expression in the mouse skin, and triggered a CD8+ T-cell response characterized by the peptide-specific secretion of IFN-γ and IL-2, but no specific antibodies. Intramuscular gene delivery resulted in a several-fold higher Luc expression and anti-Luc antibody, but induced low IL-2 and virtually no specific IFN-γ. Photon flux from the sites of Luc gene injection was inversely proportional to the immune response against GFQSMYTFV (p<0.05). Thus, BLI permitted to control the accuracy of gene delivery and transfection with respect to the injection site as well as the parameters of electroporation. Further, it confirmed the critical role of the site of DNA administration for the type and magnitude of the vaccine-specific immune response. This argues for the use of luminescent reporters in the preclinical gene vaccine tests to monitor both gene delivery and the immune response development in live animals.