Tattooing trumps intramuscular delivery for DNA vaccination

That was the hypotethesis successfully explored by researchers at the German Cancer Research Centre (Krebsforschungszentrum) in Heidelberg and the Institute of Haematology and Blood Transfusion in Prague, Czech Republic. Noting that DNA vaccines delivered through tattooing have been shown to induce better specific humoral and cellular immune responses than DNA administered by intramuscular injection, Martin Müller and colleagues compared DNA immunisation protocols using different routes of administration (intradermal tattoo versus intramuscular injection) and different molecular adjuvants (cardiotoxin pretreatment versus GM-CSF DNA co-delivery) in a mouse model. Their findings were reported in the online open access journal Genetic Vaccines and Therapy​. The researchers used a codon-modified gene encoding the L1 major capsid protein of human papillovirus type 16 (HPV16) as a model antigen. This had proved to be highly immunogenic in previous experiments using intramuscular administration of DNA in combination with cardiotoxin pretreatment. Two different types of plasmid -pUF3L1h and pBSC/GM-CSF respectively - were used for the induction of antigen-specific immune responses and as an adjuvant in the DNA immunisation experiment. The anaesthetised mice were immunised with DNA four times, each receiving 50 micrograms of plasmid pUF3L1h (six groups) or pBSC/GM-CSF (control group) in a single immunisation dose. Two groups of mice were given a mixture of 50 micrograms pUF3L1h DNA and 50 micrograms pBSC/GM-CSF DNA per animal in a single dose. The tattooed DNA was delivered in a 10 microlitre TE (Tween 80 and diethyl ether) buffer for single plasmid administration or a 20 microlitre TE buffer for the mixtire of plasmids in one or two drops to the shaved skin, followed by tattooing with a 7-linear needle using a commercial tattoo machine. The procedure was well tolerated, although local trauma with minor swelling or reddening of the skin was observed. In addition, some mice were pretreated with 50 microlitres of cardiotoxin five days before the first DNA immunisation. Blood was then collected from the immunised mice 10 days after the third and nine days after the fourth DNA immunisation. An antigen capture ELISA (enzyme-linked immunosorbent) assay was used for detection and endpoint titration assays of HPV 16 L1-specific antibodies, and an ELISPOT assay to measure L1-specific cellular immune responses. The researchers found that cardiotoxin pretreatment or GM-CSF DNA co-delivery substantially enhanced the efficacy of DNA vaccine delivered via the intramuscular route but had virtually no effect on the intradermal tattoo vaccination. The effect of both adjuvants was more pronounced after three rather than four immunisations. However, three immunisations without an adjuvant induced significantly higher L1-specific humoral immune responses than three or even four intramuscular DNA injections supported by molecular adjuvants. Tattooing also elicited significantly higher L1-specific cellular immune responses than DNA combined with adjuvants and delivered intramuscularly. Intramuscular administration of DNA by simple injection "is considered to be one of the less effective routes of DNA vaccination​", the researchers point out. Among commonly used methods, the best efficacy to date has been achieved following in vivo​ electroporation and gene gun delivery. Modified tattooing devices have been used in medical research to deliver various materials to the skin for different purposes (e.g., bleomycin for the treatment of hypertrophic scars, DNA for prospective gene therapy of skin disorders or vaccination), while techniques based on multiple puncturing are employed in human medicine to assess immune responses and for vaccination. As tattooing involves a much larger area of the skin than intradermal injection, Müller et al note, it has the advantage of potentially transfecting more cells. Although the researchers did not determine the mechanisms by which DNA tattooing in the mouse model induced a superior immune response to intramuscular delivery, they speculated that the effect might be due to: (a) better uptake of the DNA by non-antigen-presenting cells; (b) better uptake of the DNA by antigen-presenting cells; (c) the duration of expression; (d) the induced traumata accompanying the tattooing (i.e., causing sufficient inflammation to prime the immune system). Tattooing has the added benefits of low cost and a standardised method of application, the researchers observe. The downsides are that the procedure is "somewhat cumbersome​" and the local traumata induced "might not be considered acceptable in routine prophylactic vaccination settings involving human subjects​" - in other words, it is likely to be painful. All the same, Müller et al conclude, DNA vaccination via tattoo "seems to be the method of choice if faster and stronger immune responses have to be achieved​". Potential applications might include prophylactic vaccination of livestock or delivering therapeutic vaccines to humans, they suggest.