To Explore The Immunotherapy Effect And Epitope Vaccine Of Major Allergen Group 3 From Dermatophagoides Farina

Objective: To explore the immunotherapy effect and epitope vaccine of major allergen group 3 from Dermatophagoides farina.Methods: 1. Forty BALB/c mice were randomly divided into 4 groups, namely PBS group(negative control group), ovalbumin(OVA) group(positive control group), Der f 3 allergen for sensitization group(asthma group) and Der f 3 for specific immunotherapy(SIT group). The mice in asthma group and SIT group were injected intraperitoneally with dust mite extract on 0, 7 and 14 days, and inhalated nasally from day 21 for 7 days. During the 25 d – 27 d, the mice in SIT group were injected subcutaneously with Der f 3 allergen for SIT for 30 min before nasal inhalation; whereas PBS and OVA groups were treated with PBS and OVA respectively. Twenty four hours after the last challenge, all mice were sacrificed, the bronchoalveolar lavage fluid(BALF) was collected and calculated total white blood cells and eosinophils. The levels of IL-5 and IFN-γ in the BALF and the supernatant of splenocyte cultures(SSCS) were detected by ELISA, as well as the levels of specific Ig E and Ig G2 a in the sera. The lung tissues were also analyzed by haematoxylin and eosin(H&E) staining. 2. The T cell epitopes of Der f 3 allergen were analyzed through the sequence analysis using bioinformatics online tools. The five predicted peptides of T-cell epitopes were artificially synthesized. The spleen lymphocytes were co-cultured with the five T cell epitopes by using the modified MTT method and the levels of IL-2, IFN-γ, IL-4 and IL-5 in the supernatant of the cultures were detected by ELISA. 3. The linear B-cellepitopes of Der f 3 allergen were analyzed through the protein sequence by online bioinformatics. The eight predicted peptides of linear B-cell epitopes were artificially synthesized and fixed on the nitrocellulose membranes. The membranes were incubated with three allergic serum pools(4 serum samples in each), which were consisted of total 12 serum samples from the allergic individuals, and then probed with HRP-conjugated anti-human Ig E. Finally, the membranes were visualized using 3, 3’-diaminobenzidine(DAB) as substrate, and the positive spots were also measured. 4. T cell epitopes and B cell epitopes of Der f 3 which were identified by experiment of immunology in our previous work were chosen to design an artificial polypeptide(named Der f 3-peptides). After construction of a DNA encoding these peptides in order, Der f 3-peptides was expressed in Escherichia coli. The T cell lines(generated from peripheral blood mononuclear cells of Der f 3 skinprick test-positive allergic patients) were examined for T cell proliferative capacity and cytokine production upon stimulation with the Der f 3-peptides. Meanwhile, the capacity of Ig E-binding with Der f 3-peptides were compared with that of Der f 3.Results: 1. Compared with asthma group, the lung allergic inflammation changes in SIT group were significantly alleviated. The number of total cells and eosinophils in SIT group greatly decreased(p < 0.01). The level of IL-5 released from BALF and SSCS in the SIT group significantly decreased, compared with that in the OVA group and asthma group(p < 0.01); conversely, the level of IFN-γ in the SIT group was significantly higher(p < 0.01). The lower level of allergen-specific Ig E and higher level of allergen-specific Ig G2 a in the SIT group obviously observed, compared with those in the OVA group and asthma group(p < 0.01). 2. Five T cell epitopes of Der f 3 were predicted and three of which could promote the proliferation of the mouse spleen lymphocytes. The secretions of IL-2 and IFN-γ were significantly induced and the secretions of IL-4 and IL-5 were significantly decreasedby three of five prediction epitopes of Der f 3: 37DCPYQISLQSSSHFCGG54、98IYQHENYDSMTIDNDVALIKLKTPMT123and164SELQRVDIDVVSREQCDQLYS184. 3. Eight B-cell epitopes from Der f 3 were predicted successfully. Five of eight B-cell epitopes were identified with strong Ig E-binding abilities followed by specific Ig E assay. The amino acid sequences of them were following: 33KAKAGDCP40, 86 HASGGEKIQ VAEIYQHENYDSMTID110, 118LKTPMTLDQTNAKPVPLPPQGSDVKVG144, 156 QEG SYSLP163 and 199DVANGGVDSCQGDSGGPVVD218. 4. Small molecular allergen containing T cell epitopes and B cell epitopes was constructed successfully(Der f 3-peptides), which were recognized by the T cell clones and PBMC from allergic patients. Der f 3-peptides enhanced T cell proliferation, increased IL-2/IFN-γ secretion and decreased IL-4 and IL-5 production after stimulation with Der f 3-peptides compared with that of Der f 3. As a result, the capacity of Ig E-binding with Der f 3-peptides was decreased significantly compared with that of Der f 3.Conclusion: The results indicated that Der f 3 allergen can be used for SIT effectively, Three T-cell epitopes and five linear B-cell epitopes of Der f 3 allergen have been identified successfully. The low-toxic allergens with small molecule which connected T cell epitopes and B cell epitopes developed successfully, This result will provide a basis of the diagnosis and treatment for asthma.