Alternaria B-cell Mimotopes Immunotherapy Ameliorates Allergic Responses In A Mouse Model

Background: Alternaria is one of the most common molds associated with allergic diseases.Exposure to high levels of atmospheric Alternaria spores is a risk factor for asthma attacks.Sensitization to Alternaria in early childhood is an independent risk factor for asthma.Allergen immunotherapy(AIT)is still the only disease-modifying therapy for allergic diseases.High-quality allergen vaccine is one of the most important factors for the efficacy of immunotherapy.However,most of the extracts of Alternaria alternata used in clinic are derived from natural allergens,and their components are complex,which directly affects the efficacy and safety of immunotherapy.In addition,due to the variability of strains,culture conditions and extraction steps,it is very difficult to standardize the extracts of Alternaria alternata.Objective: To screen and synthesize B cell epitope mimotope vaccine of Alternaria alternata by phage display peptide technology,and evaluate the therapeutic effect of B cell epitope mimotope vaccine of Alternaria alternata on airway inflammation in Balb/c mouse asthma model.Methods: 1.Phages were incubated with the healthy human serum three times.The non-specific binding part of the phage library to the specific IgE hypervariable region of Alternaria alternata was screened out,and then were incubated three times with the serum pool of patients allergic to Alternaria alternata to screen B cell mimic epitopes that can specifically bind the specific IgE hypervariable region of Alternaria alternata.2.DNA sequencing of the mimic epitopes was carried out after amplification and incubation and matched with the three-dimensional structure of the components of Alternaria alternata.The mimic epitopes of Alternaria alternata with high matching ability were screened to synthesize mimotope vaccine.The vaccine was then combined with specific IgE in serum of patients for binding and inhibition ability.Eosinophil inhibition test was used to test its activity to eosinopils.3.Establish a mouse asthma model.Six-week-old female Balb/c mice were randomly divided into five groups: blank control group,model group,irrelevant vaccine group,Alternaria alternata extract immunotherapy group and mimotope vaccine immunotherapy group,with 6 mice in each group.On the 4th,5th and 6th day,mice were sensitized by intranasal drip of Alternaria alternata extract.The blank group was given 0.9% saline.On the 18 th,19th and 20 th day,Alternaria alternata was used to challenge,on the 36 th,37th and 38 th days,and 0.9% saline was used in the blank group.On the 21 st and 22 nd days,tail blood was taken to verify the binding ability of mimotope vaccine to serum IgE in asthmatic mice.On the 23 rd,24th,25 th and 26 th day,the mice were subcutaneously injected with 0.9% saline,irrelevant peptide vaccine,and Alternaria alternata extract and mimotope vaccine.The mice were killed within 48 hours of the last challenge.Result: 1.Compared with the model group and irrelevant vaccine group the symptoms of the mice in the mimotope vaccine group were significantly improved in the symptoms such as hair disorder,restlessness or too quiet,nose scratching,runny nose,nodding breathing,accelerated breathing and so on.2.Compared with model group and irrelevant vaccine group,the number of inflammatory cells and eosinophils in mice treated withmimotope vaccine decreased.3.Compared with model group and irrelevant vaccine group,the total IgE and specific IgE in serum of mice treated with mimotope vaccine decreased,while the total IgG1 and specific IgG1 increased.4.Compared with model group and irrelevant vaccine group,IFN-gamma increased and IL-4 decreased in alveolar lavage fluid in mice treated with mimotope vaccine.5.Compared with model group and irrelevant vaccine group,the proportion of Th1/Th2 cells in alveolar lavage fluid of mice treated with mimotope vaccine increased.6.The infiltration of alveolar and bronchial inflammation was significantly reduced and airway stenosis was improved in the mimotope vaccine treatment group.Conclusion: The results show that the B cell mimic peptide vaccine of Alternaria alternata can improve the inflammatory allergic reaction of asthmatic mice and has potential as a new drug for specific immunotherapy of IgE-mediated allergic diseases of Alternaria alternata.