Identification Of Linear IgE-binding Epitopes Of Artemisia Annua Major Allergen And Determination Of Pollen Sensitization Of Artemisia Ordosica

Background:Type Ⅰ hypersensitivity reaction is an allergic disease that involves mast cells,basophils,and other effector cells that release inflammatory mediators.IgE antibodies play a pivotal role in the development and regulation of allergic responses and are directly responsible for the allergic cascade.Studies have shown that pollen produced by Artemisia plants is one of the top ten airborne allergens in the world and is also the most important outdoor allergen source in China,which can cause rhinitis and asthma in allergic patients.As a traditional Chinese medicine,Artemisia annua(A.annua)has been widely used to treat various diseases.However,pollen grains from A.annua are an important cause of allergic disease during late summer and fall in northern China.Indepth studies of IgE-binding epitopes of allergens are important for the development of novel diagnostic and therapeutic approaches.Art an 1 and Art an 3 are two major allergens of A.annua,but related studies are very limited,and their IgE-binding epitopes have not been reported so far.Studies have revealed a close connection between the rise in the planting area of the sand-fixing Artemisia ordosica(A.ordosica)in northwest China and the rise in the prevalence of allergic diseases there.However,there is no concrete research verifying A.ordosica as the major cause of seasonal allergy in those areas.Objectives:1.To identify the linear IgE binding epitopes of Art an 1 and Art an 3,the two major allergens in A.annua pollen,evaluate the immunogenicity of epitope peptides,and to investigate their potential use as a vaccine component to prevent A.annua pollen allergic disease.2.To examine the protein-coding information of the major allergens in A.ordosica pollen and compare the protein homology with other major Artemisia species.To evaluate the allergenicity of A.ordosica pollen extract,so as to establish the relationship between A.ordosica pollen and the outbreak of seasonal allergic diseases in the A.ordosica habitat areas.Methods:1.The major allergens of A.annua(Art an 1、Art an 2 and Art an 3)were expressed in E coli.rArt an 3 was purified by Ni column affinity chromatography and gel filtration chromatography,and its IgE reactivity was evaluated by immunoblot assay.Overlapping peptides covering full-length Art an 1 and Art an 3 proteins were synthesized and the linear IgE binding epitopes of Art an 1 and Art an 3 were characterized by dot-blot assay.CRM 197 was used as a carrier protein and was coupled with individual epitope peptide.The specific IgG antibody levels induced by immunizing the mice with carrier-coupled Art an 3 peptides were measured by ELISA.The inhibition of human IgE binding to rArt an 3 by peptide-specific mouse antibodies was analyzed by ELISA competition assay.Art an 3 homology model was built based on the Art v 3 crystal structure and its epitope peptide regions were mapped.2.Transcriptome sequencing of Artemisia ordosica flower and pollen was conducted and homolog of Art an 1 and Art an 3 was identified.Multi-sequence alignment was performed to study the homology with six other Artemisia species.The allergic airway inflammation mice model induced by A.ordosica pollen extract was established.Serum total IgE antibody levels and pollen-specific IgE antibody levels,as well as IL-4、IL-5 and IFN-γ in BALF were measured by ELISA.Mouse lung pathological sections were made to observe the inflammatory changes.Results:1.Art an 1、Art an 2 and Art an 3 could be successfully expressed in soluble form when fused with trigger factor.The recombinant Art an 3 was purified in homogeneity and its IgE reactivity was verified.Dot-blot results showed that there are two major IgE linear epitope regions in both Art an 1 and Art an 3.When conjugated to CRM197 and immunized mice,both of the Art an 3 B-cell epitope peptides induced Art an 3-specific IgG antibodies,which could inhibit Artemisia allergic patients’ serum IgE binding to Art an 3.The IgE-binding epitopes were well-exposed on the protein surface as illustrated by three-dimensional structure model of Art an 3.2.Transcriptome sequencing results showed that the two major allergens of A.ordosica share a high identity with their counterparts of six Artemisia species at the amino acid level.The allergic airway inflammation mice model showed that the levels of IL-4 and IL-5 in BALF were increased and IFN-y was decreased,the levels of total IgE antibody and pollen-specific IgE antibody were significantly increased.In addition,severe pathological changes also occurred in the lung tissue of the allergic mice.These results indicated that the A.ordosica pollen extracts could induce allergic responses in mice and the allergenicity of A.ordosica pollen was elucidated for the first time.Conclusions:1.This research demonstrated that there are two linear IgE binding epitope regions in both Art an 1 and Art an 3 and successfully identified two linear IgE binding epitope peptides of Art an 3.2.The two major allergens of A.ordosica identified by transcriptome sequencing share a high identity with other Artemisia species.The allergenicity of A.ordosica pollen extracts was verified by an allergic airway inflammation mice model.