Study On Immunotherapy Of Dust Mite Allergic Asthma With TLR7Agonist Adjuvant

Allergic asthma is a chronic disease characterized by airway inflammationcaused by the disregulated production of cytokines secreted by allergen-specific type2T helper (Th2) cells. It has been documented that Toll like receptor7(TLR7)stimulation triggers not only antiviral responses, but also alleviates experimentalallergic asthma.In the present study，we used a new synthesized versatile TLR7agonist, conjugated it to recombinant Der f1, a dust mite antigen，to conduct theimmunotherapy on a mouse model of dust mite-sensitized allergic asthma. Theimmunomodulatory effects of the new vaccine and its molecular mechanisms wereinvestigated.Methods: First of all, we established TLR7a-Der f1vaccine.The TLR7a-Der f1conjugate was identified by a UV absorption peak at280nm. SDS-PAGE and circulardichroism analysised whether protein structure was changed or not. Theimmunogenicity and immunoantigenicity were identified respectively byWestern-blot and ELISA. Then we used the dust mites crude extracts sensitized miceto establish allergic asthma model. Following the treatment, airwayhyperresponsibility(AHR) was investigated immediately. Mouse serum was used todetect the specific IgE, IgG2a and IgG1antibodies levels. Bronchoalveolar lavagefluid (BALF) and spleen cell culture supernatant were collected to measure theexpression of several Th1/Th2-type cytokines(IL-4, IL-10, IFN-γ and IL-12). And thepathological changes of lung tissue were observed.Results: We successfully established TLR7a-Der f1vaccine. TLR7a wascovalently coupled to Der f1at a5:1molar ratio. The primary structure andsecondary structure of TLR7a-Der f1did not change a lot after coupled. TheTLR7a-Der f1vaccine still had potential capability of immunogenicity andimmunoantigenicity that laid foundation for the next phase of immunotherapy. Micetreated with Der f1and TLR7a-Der f1vaccine prior to challenge displayed alleviatedsymptoms such as airway hyperreactivity, lung inflammation and less eosinophilic cells in bronchoalveolar lavage fluid. In addition, reduced responses of Der f-specificIgE and increased responses of Der f-specific IgG2a and IgG1were aroused in theDer f1and TLR7a-Der f1vaccine group. Although both Der f1and TLR7a-Der f1has inhibited effect on allergic asthma, the efficiency of TLR7a-Der f1to bebetter.We also observed that IL-4was reduced and IFN-γ and IL-12were increasedamong splenocytes and in bronchoalveolar lavage fluid in the TLR7a-Der f1vaccinegroup. However, TLR7a-Der f1could not induce IL-10secretion as Der f1does.TLR7a and Der f1mixture vaccine also had a therapeutic effect on allergic asthma,but the effect is not obvious compare with coupling TLR7a to Der f1.Theanti-inflammatory effect was significantly lower after blocking TLR7withchloroquine. Mice treated with TLR7a and chloroquine developed allergic asthma.Conclusion: Our results illustrate that TLR7a-Der f1vaccine may play a role inimmunologic protection in murine allergic asthma. The potential mechanism ispossibly by directly inducing Th2reaction switch to Th1reaction.The vaccine isexpected to become a new approach to the treatment of allergic diseases.