Supplementary Material for: Construction of a hybrid vaccine based on Der f 35-derived peptides with reduced allergenicity

Introduction
House dust mite is the primary trigger of allergic respiratory diseases worldwide, and allergen-specific immunotherapy (AIT) is the only disease-modifying treatment in clinic. The use of allergen molecules instead of extracts is a promising strategy in AIT. In the study, we constructed a peptide hybrid vaccine against the major mite allergen Der f 35 and verified its hypo-allergenicity to be a promising candidate for AIT of mite allergy.
Methods
The gene encoding Der f 35 was retrieved and synthesized. The hypoallergenic peptide fragments derived from the B-cell epitopes were synthesized based on the predicted profiles of B-cell or helper T cell epitopes in Der f 35, they were verified by Immunoglobulin E (IgE)-reaction test and fused to non-allergenic protein carrier to form the hybrid vaccine. Both the wild-type Der f 35 and the designed vaccine were expressed in Escherichia coli and purified by chromatography, their IgE-binding activity was compared by indirect enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA and basophil activation test (BAT). The blocking immunoglobulin G (IgG) against the designed vaccine was raised in rabbits and its ability to inhibited IgE-binding of Der f 35 was evaluated by ELISA. The vaccine's effects on peripheral blood mononuclear cells (PBMCs) were investigated.
Results
A total of 29 out of 60 (48.33%) IgE-positive sera against Der f 35 were screened. Five peptide fragments (residue 39-42, 60-67, 73-107, 111-118, 126-143) from Der f 35 were selected as candidates, in which four peptides exhibited almost no IgE-reactivity and the fragment 73-107 had weak reaction. Only 5.98-24.02% inhibition rates could be achieved by the peptides when compared with Der f 35 (97.07%). The designed vaccine migrated at approximately 30 kDa by SDS-PAGE. The IgE-ELISA revealed a significant reduction in IgE-binding activity to the vaccine when compared to wild-type Der f 35 (p < 0.0001), the decreased allergenicity were further confirmed by IgE-Western blot, inhibition ELISA and BAT, respectively. The IgE-reactivity of Der f 35 could be blocked by the vaccine induced IgG (p < 0.01). The levels of IL-5 and IL-13 from PBMCs were significantly decreased after stimulation by the vaccine than that by Der f 35 (p < 0.05).
Conclusion
The designed B-cell epitope vaccine of Der f 35 showed greatly diminished allergenicity and Th2 activity. It could be an effective and safety candidate to prevent the allergic adverse reactions during the immunotherapy of mite allergy and merits the further study.