2.4∣Experimental design of therapeutic and prophylactic DNA vaccination by epicutaneous application
Female 6-week-old BALB/c mice were purchased from the National Laboratory Animal Center, Taiwan and raised under specific pathogen-free conditions. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Taichung Veterans General Hospital (approval no. La-1081655). The mouse model of F. taiwanaallergy obtained by sensitization to the major allergen, For t 2, was established as previously described.20,22
The pilot dose-finding schedule is summarized in Supplementary Figure S1. Groups of mice (n=6) were first sensitized with 2 intraperitoneal injections (IP) of 10 μg rFor t 2 absorbed by 2 mg alum adjuvant, with a 1-week interval between each injection. Topical delivery of DNA antigens was performed as follows. To elicit a specific immune response, on day 13, the hair of the abdominal area of the mice was removed using a depilatory (Supplementary Figure S2). In addition, tape stripping was used to disrupt the skin barrier at days 14, 21, and 28 before epicutaneous DNA vaccination. The three groups of skin-vaccinated mice (SV10, SV25 and SV50) received the For t 2 DNA vaccine three times using a volume of 10-50 μl administered to the pre-shaved skin at doses of 10, 25, and 50 μg for 1 hour, respectively. The non-vaccinated (sham) group served as the negative control and received a topical application of the same volume of PBS alone. All groups of mice were challenged intradermally (ID) with 3 doses of rFor t 2 for 3 consecutive days between days 59-61. Blood samples were collected bi-weekly from the submandibular vein. The scratching behaviors of the mice were video recorded on days 0 and 61, and the mice were sacrificed on day 63.
Next, we investigated the effect of 25 μg For t 2 DNA/patch on the treatment and prevention of biting midge allergy. Timelines of the therapeutic and prophylactic vaccination and the grouping of experimental mice are summarized in Figure 1A, 1B and 1C, respectively. For the therapeutic vaccination, three treatments using skin patches containing the DNA vaccine were given after sensitization to For t 2 (Figure 1A). For the prophylactic approach, mice received three DNA patches prior to sensitization with recombinant For t 2 (Figure 1B).