Allergy Asthma Immunol Res.  2016 Jan;8(1):55-62. 10.4168/aair.2016.8.1.55.

Effect of Amino Acid Polymorphisms of House Dust Mite Der p 2 Variants on Allergic Sensitization

• Tanyaratsrisakul S
• Jirapongsananuruk O
• Kulwanich
• Hales BJ
• Thomas WR
• Piboonpocanun S

• KMID: 2166650
• DOI: http://doi.org/10.4168/aair.2016.8.1.55

Abstract

PURPOSE
The sequence variations of the Der p 2 allergen of Dermatophagoides pteronyssinus diverge along 2 pathways with particular amino acid substitutions at positions 40,47,111, and 114. The environmental prevalence and IgE binding to Der p 2 variants differ among regions. To compare IgE binding to Der p 2 variants between sera from Bangkok, Thailand and Perth, Western Australia with different variants and to determine the variant-specificity of antibodies induced by vaccination with recombinant variants.
METHODS
The structures of recombinant variants produced in yeast were compared by circular dichroism and 1-anilinonaphthalene 8-sulfonic acid staining of their lipid-binding cavity. Sera from subjects in Bangkok and Perth where different variants are found were compared by the affinity (IC50) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2.0109 was measured.
RESULTS
The secondary structures of the recombinant variants resembled the natural allergen but with differences in ANS binding. The IC50 of Der p 2.0101 required 7-fold higher concentrations to inhibit IgE binding to the high-IgE-binding Der p 2.0104 than for homologous inhibition in sera from Bangkok where it is absent, while in sera from Perth that have both variants the IC50 was the same and low. Reciprocal results were obtained for Der p 2.0110 not found in Perth. Direct binding revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to other variants.
CONCLUSIONS
The affinity of IgE antibody cross-reactivity, the direct IgE binding, and the specificities of antibodies induced by vaccination show that measures of allergic sensitization and therapeutic strategies could be optimized with knowledge of Der p 2 variants.

Keyword

Allergic reaction; Der p 2 Allergen; IgE binding

MeSH Terms

Amino Acid Substitution
Animals
Antibodies
Circular Dichroism
Dermatophagoides pteronyssinus
Dust*
Hypersensitivity
Immunoglobulin E
Immunoglobulin G
Inhibitory Concentration 50
Mice
Prevalence
Pyroglyphidae*
Thailand
Vaccination
Western Australia
Yeasts
Antibodies
Dust
Immunoglobulin E
Immunoglobulin G

Figure

• 

  Fig. 1 Hydrophobic binding to Der p 2 variants by ANS staining. The data show λmax of emitted fluorescent intensity value from ANS-stained hydrophobic cavity of isoforms.

• 

  Fig. 2 IgE binding affinities of rDer p 2 variants as determined by IC50. The ability of different variants to inhibit IgE binding to rDer p 2.0104 subjects from (A). Bangkok (n=7) and (B) Subjects from Perth (n=9) was titrated. Results show the mean (SE) of inhibition at different concentrations from 5-7 determinations with the IC50 calculations in the inserted tables.

• 

  Fig. 3 IgE binding of sera from 2 populations to rDer p 2variants. To account for variation in the titer of different individuals to Der p 2, the results were normalized by expressing them as a ratio of titer to Der p 2.0101: titer to each variant IgE (n=7 Perth and n=8 Bangkok). Statistical analysis was done by one way ANOVA, *P<0.05, **P< 0.01, ***P<0.001. The difference calculated for a ratio of titer to Der p 2.0101/0104 were also calculated with difference compared to that of Der p 2.0101/0103, 0109, and 0110 being significant in the Perth sera (P<0.001, P<0.001, and P<0.01, respectively). Also a ratio of titer to Der p 2.0101/0103 and 0101/0109 of the 2 regions are significantly different (P<0.05).

• 

  Fig. 4 Inhibition of IgG binding by polyclonal mouse anti-rDer p 2.0101antibodies to Der p 2.0101 by rDer p 2 variants. The data show an average percent inhibition of mouse anti-IgG against 2.0101 with SE from 3-5 experiments. The IC50 values for the variants are shown in the inserted table.

• 

  Fig. 5 Direct binding of mouse anti-Der p 2.0101. (A) anti-Der p 2.0110 (B) IgG antibodies to rDer p 2 variants. Assays were performed 7 times. The results were normalized by expressing them as percentage of a mean average value of IgG binding to 2.0101(A) or 2.0110 (B) that of IgG binding to variants.

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