Allergy Asthma Immunol Res.  2016 Mar;8(2):124-131. 10.4168/aair.2016.8.2.124.

Mimotopes for Api g 5, a Relevant Cross-reactive Allergen, in the Celery-Mugwort-Birch-Spice Syndrome

• Lukschal A
• Wallmann J
• Bublin M
• Hofstetter G
• Mothes-Luksch N
• Breiteneder H
• Pali-Scholl I
• Jensen-Jarolim E

• KMID: 2165928
• DOI: http://doi.org/10.4168/aair.2016.8.2.124

Abstract

PURPOSE
In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome.
METHODS
Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined.
RESULTS
Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope.
CONCLUSIONS
The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.

Keyword

Celery-mugwort-birch-spice syndrome; IgE Epitopes; food hypersensitivity; high molecular weight (HMW) allergens; mimotope; vaccination

MeSH Terms

Allergens
Animals
Apiaceae
Apium graveolens
Artemisia
Bacteriophages
Betula
Carbohydrates
Clone Cells
Enzyme-Linked Immunosorbent Assay
Food Hypersensitivity
Glycoproteins
Horseradish Peroxidase
Humans
Immune Sera
Immunization
Immunoglobulin E
Immunoglobulin G
Mice
Molecular Weight
Pollen
Spices
Vaccination
Virtues
Allergens
Carbohydrates
Glycoproteins
Horseradish Peroxidase
Immune Sera
Immunoglobulin E
Immunoglobulin G

Figure

• 

  Fig. 1 Binding characteristics of the monoclonal antibody BIP3 on birch pollen extract and cross-reactive allergens Panel A BIP 3 recognizes higher molecular weight proteins in blotted birch pollen extract, which, to a great part, is abrogated after periodate deglycosylation. Lanes n, the untreated extracts; d, birch pollen extract after periodate treatment. Panel B Monoclonal antibodies BIP3 and BIP 1 recognize epitopes in blotted birch pollen extract and cross-reactive allergens of relevance in the celery-mugwort-birch-spice syndrome. BIP1 (lane 1) recognizes Bet v 1 and homologues; BIP3 (lane 3) shows binding to HMW allergens in celery, birch and mugwort pollens. Lanes C, buffer controls.

• 

  Fig. 2 Increasing phage titers during biopanning indicate enrichment of specific phages; the deduced amino acid sequences of identified phage clones are depicted in a phylogenetic treePanel A: The number of BIP3-eluted phages increased during biopanning from 2.4×104 in the first round to 6.2×107 in the fourth round. Panel B: Phylogenetic tree indicating the degree of similarity between the selected peptides. Clones used for further analysis are marked by *

• 

  Fig. 3 Selected mimotope clones inhibit human IgE binding to birch pollen HMW allergen. A birch pollen reactive human serum was used either untreated or pre-incubated using 107 phage particles before application to blotted birch pollen extract. Lane 1, no pre-incubation; lane 2, 1-12-cyclo-CKASSCDTGHC; lane 3, 1-12-cylo-CFFAWRSLPNC; lane 4, 1-6-cyclo-CHKLRCDKAIA; lane 5, phage of unrelated specificity, (CRQTRTRTMPGCG); lane 6, original phage library; lane 7, helper phage.

• 

  Fig. 4 Immunizations with phage-displayed mimotopes induce BIP3-type IgG in BALB/c micePanel A: Immunization schedule for the 3 selected phage clones. Panel B: IgG reactivities of 3 mice of each group immunized with different BIP3-specific phage clones on blotted birch pollen extract. Group 1, mimotope sequence CKASSCDTGHC; group 2, CFFAWRSLPNC; group 3, CHKLRCDKAIA. Lane 0, pre-immune serum; lane 1, the first immune serum from day 21; lane 2, the second immune serum from day 38.

• 

  Fig. 5 Identification of Api g 5 as the BIP3 target antigen by mimotope induced immune sera and titer determination. (A) The IgG reactivity of pre-immune (PIS) and the second immune sera (MIS) of mice immunized with the dominant BIP3 mimotope clone CHKLRCDKAIA is shown. y-axis indicating relative binding intensity in ELISA coated with different antigens: glycosylated celery allergen Api g 5 (black bars), non-glycosylated grass pollen allergen Phl p 5 (grey), and weak reactivity to model-glycoprotein HRP (white). (B) Sera of the mice were pooled at equal parts and diluted as indicated on the x-axis before testing on coated Api g 5 in ELISA. Bound IgG (black columns, immune sera; white columns, pre-immune sera) was detected by HRP-labeled anti-mouse IgG. The y-axis indicates the OD values determined at 405 nm.

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