A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

• Published in: PloS one 2015-06, Vol.10 (6), p.e0130375-e0130375
• Main Authors: Kinnear, Ekaterina, Caproni, Lisa J, Tregoning, John S
• Format: Article
• Language: English
• Subjects: Animals; Antigens; CD8 antigen; CHO Cells; Clinical trials; Coding; Comparative analysis; Cricetinae; Cricetulus; Deoxyribonucleic acid; DNA; DNA vaccines; Dogs; Electroporation; Female; Fluorescence; Gene Expression; Hemagglutinins; Humans; Imaging; Immune response; Immune system; Immunization; Immunogenicity; Infection; Infections; Influenza; Influenza A Virus, H1N1 Subtype - immunology; Influenza Vaccines - genetics; Influenza Vaccines - immunology; Influenza Vaccines - metabolism; Influenza, Human - prevention & control; Luciferase; Luminescent Proteins - biosynthesis; Luminescent Proteins - genetics; Madin Darby Canine Kidney Cells; Medical research; Mice, Inbred BALB C; Pathogens; Plasmids; Proteins; Red Fluorescent Protein; Swine influenza; Tattoos; Transfection; Vaccine efficacy; Vaccines; Vaccines, DNA - genetics; Vaccines, DNA - immunology; Vaccines, DNA - metabolism; Virology; Whole Body Imaging
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0130375

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.