Backgrounds: Allergen-specific immunotherapy (ASIT) is capable of inducing immune tolerance to the corresponding allergen, thereby enabling treatment of the root cause of the allergic disease. 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 during the COVID-19 pandemic. Methods: We established a mouse model allergic to the biting midge, Forcipomyia taiwana, to test the concept of the epicutaneous DNA vaccine. The experiments were designed using two approaches: therapeutic and prophylactic. Mice were patched with 25 μg For t 2 DNA vaccine patches for one hour with a total of three treatments spaced one week apart. Scratch bouts after For t 2 allergen challenge were used as a clinical surrogate of itch and biomarkers for allergic inflammation were examined by ELISA, RT-PCR, and histopathology. Results: We found that after epicutaneous DNA vaccination, the mice significantly improved with respect to allergen-induced scratch. For t 2-specific IgE, mRNA, and protein of IL-13 and eosinophils infiltration in the targeted skin decreased. For t 2-specific -IgG2a and mRNA expression of FOXP3 increased. Conclusions: We demonstrated the first needle-free epicutaneous DNA vaccine patch that is effective in preventing as well as treating biting midge allergy in a murine model. The mice showed improvements in their allergic condition both clinically as well as in allergic inflammation. The mode of this anti-allergic DNA vaccine may have the potential for use in other specific immunotherapies for other allergens.

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.2The 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. For t 2 is the most predominant,with 75% of midge-allergic patients showing specific IgE to For t 2.4 Allergic reactions to midge bites are not limited to humans but also seen in livestock, such as horses, cattle, sheep, and donkeys, causing significant veterinarian problems.E.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). The therapeutic approach is to imitate ASIT in human with established allergy while the prophylactic approach to non-allergics. Twenty-five μg For t 2 DNA was determined as the optimal dose after dose-finding experiments (Supplementary Fig S1). 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 hoursand had significantly increased at 48 hours after last treatment (Fig S3). Scratch bouts after rFor t 2 challenge were used as a clinical surrogate of itch. We measured For t 2-specific IgE, IgG1 and 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.After epicutaneous DNA vaccination, the allergen-induced itchin both groups significantly improved, and For t 2-specific IgE and IgG1/IgG2a ratio decreased significantly at week 6 or week 8 (Fig 2). Levels of mRNA and protein of IL-13 decreased significantly, but IFN-gamma and IL-10 remained unchanged. Expression of FOXP3 mRNA increased (Fig 2, protein data not shown). Eosinophils infiltration in the challenged skin significantly decreased (Fig S4).This is the first study to demonstrate an epicutaneous needless anti-allergic DNA vaccine that effectively treats an established allergic condition and prevents the development of an allergic disease using biting midge allergy as a model. After epicutaneous DNA vaccination, in addition to allergen-induced itch, the changes of multiple biomarkers suggest that immune tolerance was induced after 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 can penetrate the dermal barrier and translate the corresponding protein in the targeted skin and 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 potentially be used 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