DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in
pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to
evoke a strong immune response, possibly due to poor antigen expression. To improve
antigen expression, improved technology to monitor DNA vaccine transfection efficiency is
required. In the current study, we compared plasmid encoded tdTomato, mCherry,
Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging.
The intramuscular, subcutaneous and tattooing routes were compared and electroporation
was used to enhance expression. We observed that overall, fluorescent proteins were not a
good tool to assess expression from DNA plasmids, with a highly heterogeneous response
between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato
or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal
observed. Subcutaneous delivery was weakly visible and nothing was observed following
DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune
responses mirrored visible expression levels. A protective immune response against H1N1
influenza was induced by all routes, even after a single dose of DNA, though qualitative differences
were observed, with tattooing leading to high antibody responses and subcutaneous
DNA leading to high CD8 responses. We conclude that of the reporter proteins used,
expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the
disconnect between visible expression level and immunogenicity suggests that in vivo
whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine
efficacy.