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).