Needle-free Epicutaneous For t 2 DNA Vaccine is Effective for Preventing and Treating Biting Midge (Forcipomyia taiwana) allergy in a murine modelTo the Editor,Allergen-specific immunotherapy (ASIT) remains the only treatment capable of inducing immune tolerance to the corresponding allergen and potentially treating the root cause of the allergic disease.1 As the treatment course of protein-based vaccines for ASIT is time-consuming, an easily administered epicutaneous anti-allergic DNA-based vaccine is an attractive method, especially in light of the COVID-19 pandemic.2 We established a mouse model of biting midge allergy to test the concept of the epicutaneous DNA vaccine.The biting midge, Forcipomyia taiwana , is the most prevalent cause of biting insect allergy in Taiwan. It is a tiny hematophagous midge that attacks en masse. As many as 60% of exposed individuals develop allergic reactions to the bites.3 The midge is widely distributed throughout Taiwan and southern China. Among the identified allergens, For t 2 is the most predominant, with 75% of midge-allergic patients showing specific IgE to For t 2.4E.coli -expressed For t 2 recombinant protein (rFor t 2) was used as an allergen to sensitize and challenge the mice.5For t 2-encoding fragment (GenBank accession EU678971) was amplified by PCR. The PCR products were subcloned into pVAX1 (Life Technologies, Carlsbad, CA) . The experiments were designed using two approaches: therapeutic and prophylactic (Fig 1). Twenty-five μg For t 2 DNA was determined as the optimal dose after several dose-finding experiments (Fig S1, data not shown). For each treatment, the hair of the abdominal area of the mice was removed using a depilatory, tape-stripped, then patched with 25 μg For t 2 DNA vaccine for one hour and removed. A total of three treatments were given spaced one week apart (Fig 1 and Fig S2). For t 2 proteins were detected in the patched skin and the immune organ spleen at 24 hours and had significantly increased at 48 hours (Fig S3). Scratch bouts after rFor t 2 challenge were used as a clinical surrogate of itch. We measured total IgE and For t 2-specific IgG2a in the sera as well as mRNA and proteins of IL-13, interferon-gamma, IL-10, and FOXP3 in the culture supernatants of splenocytes after stimulation with various doses of rFor t 2 at 37℃ for 3-5 days by ELISA and real-time quantitative PCR. Histopathology of the challenged skins was examined.We found that after epicutaneous DNA vaccination, the allergen-induced itch of the mice significantly improved, and For t 2-specific IgG2a increased (Fig 2). Both mRNA and protein of IL-13, and eosinophils infiltration in the targeted skin, significantly decreased. Expression of FOXP3 mRNA increased (Fig S4-S6).This is the first study to demonstrate an epicutaneous anti-allergic DNA vaccine that is effective at both treating an established allergic condition and preventing the development of an allergic disease using biting midge allergy as a model. After epicutaneous DNA vaccination, in addition to the significant improvement in allergen-induced itch, the changes of biomarkers for allergic inflammation, including IgE, allergen-specific IgG2a, allergen-challenge-induced eosinophil infiltration in the skin, Th2 cytokines from the splenocytes, and regulatory T cell-related transcription factors, suggest that immune tolerance was induced after three patches of the epicutaneous DNA vaccine.Our data show that though the molecular weight of the For t 2 DNA vaccine is as high as 4000 base pairs, it is able to penetrate the dermal barrier and translates the corresponding protein in the targeted skin as well as the spleen of the vaccinated mice. It is possible that the DNA vaccine passes the epidermis via the hair follicles as the skin is tape-stripped before epicutaneous vaccination.6The mode of this anti-allergic epicutaneous DNA vaccine may have potential for use in other specific immunotherapies for other allergens.Mey Fann Lee1Chi Sheng Wu2 Shyh Jye Lin3Yi Hsing Chen2,4*1Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan2Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan3School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan4School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan