The antigenicity of TLR7a-Der f 1 was detected by ELISA. IL-10 from T cells, proliferation of B-lymphocytes Z-LEHD-FMK and memory B cells, production of IgG1 and IgG4 antibodies, and inhibit the activation of Th2 effector cells [12-15]. Epicutaneous immunization (EPI) with Bey Z-LEHD-FMK v 1 (the Z-LEHD-FMK major brich pollen allergen) plus R848 induced Bet v 1-specific Th1 responses and suppressed asthmatic features [16]. In this study, we synthesized a new versatile TLR7 agonist conjugated to Der f 1 and evaluated the modification of TLR7 signaling on the allergic responses elicited by HDM. Materials and methods Animals Female BALB/c mice (6-8 weeks) were purchased from the Animal Center of Guangdong Province. The mice were maintained in a specific pathogen-free facility at the Experimental Animal Center of Shenzhen University. All animal care and experimental protocols were carried in accordance with the Institutional Guidelines for Animal Care and Use of Laboratory Animals at Shenzhen University. Preparation of Der f 1 antigen The pET-28a (+) plasmid containing Der f 1 gene was transformed to the host cell BL21 as previously reported [17]. The protein was purified by Ni affinity chromatography, and used as a specific antigen. SDS-PAGE and Western blot were used to access the purification of the protein. Conjugation of TLR7 agonist to Der f 1 Succinimidyl 6-hydrazino-nicotinamide acetone hydrazone (SANH) was used as a linker to couple amino groups on proteins. Initially, TLR7a was added to Der f 1 at a ratio of 1 1:40 and shacking overnight at 15C. Uncombined agonist was removed by ultrafiltration tubes (10kDa) as previously described [18]. The conjugated TLR7a-Der f 1 was assessed by UVat 280 nm. The secondary structure of TLR7a-Der f 1 was characterized by circular dichroism (CD) [19]. The IgE-binding reactivity was measured by enzyme-linked immunosorbent assay (ELISA). Immunization protocols The antigen sensitization and challenge and immunotherapy of the murine model of allergic asthma were performed as previously described [20-22]. Briefly, BALB/c mice received immunization with 50 g of house dust mite (HDM) extract in 0.2 ml PBS with 2 mg of Al(OH)3 (Sigma, USA) (M) by intraperitoneal injection on day 0, 7 and 14. TIE1 14 days after sensitization, then treated with 100 g Der f 1 adsorbed on 2 mg of Al(OH)3 (D), 100 g TLR7a-Der f 1 (T-D) or TLR7a (T) in 0.1 ml PBS daily for 3 times. The mice were challenged with 50 g HDM antigen administered by nasal drop from days 41 to 47. Mice sensitized and challenged with normal saline were used as control group (C). Twenty-four hours after the final challenge, airway hyperreactivity (AHR) was assayed in a Buxco plethys-mograph (Buxco, USA) and next day all the mice were sacri?ced (Figure 1). Open in a separate window Figure 1 Protocols of allergic asthma sensitization, challenge and treatment. Mice were administered house dust mite (HDM) from day 0 to 14. Treatment was started from day 28 to 34, once every three days, for a total of three times. Epicutaneous applications of Der f 1+Al(OH)3 or Der f 1-TLR7a or TLR7a. After challenge with HDM for a 1-week period, the mice were challenged with Mch for AHR detection, and mice serum and BALF were collected after sacrifice. AHR measurement Airway hyperresponsiveness (AHR) to methacholine (Mch) aerosol was evaluated as an increased pulmonary resistance using unrestrained whole-body plethysmography with a four-chamber system (Buxco Research Systems, Wilmington, NC, USA) [23]. Firstly, mice were put into the chamber and kept steadily respiration for 10 min. Z-LEHD-FMK The baseline of breathing was monitored for 5 min. The records for Penh values begin with the event that inhaling 0.1 ml NS..