Insights into Api m 10-Isoforms and Splice Variants: More Than One Major IgE-Binding Epitope

Honey bee venom (HBV) often triggers severe IgE-mediated allergies. The major allergen icarapin (Api m 10) has attracted attention due to low occurrence in some HBV immunotherapy products. Despite being a major allergen, little is known about the Api m 10 structure and IgE-binding regions. This study aimed to characterize its IgE-binding epitopes and structure in more detail.

Overlapping Api m 10-specific peptides covering the sequences of the 11 known Api m 10-isoforms and variants were synthesized and spotted on microarray slides (15 amino acids (AA), off-set: 4 AA). Sera from 28 HBV-allergic patients with detectable Api m 10-specific IgE were used to characterize the distinct IgE-binding profiles to all Api m 10-variants. Sera from ten Api m 10-immunized BALB/c mice were used to investigate possible shared epitopes between humans and mice. All Api m 10-variants were investigated for secondary structural elements via circular dichroism spectroscopy and potential aggregation via dynamic light scattering.

We identified 7 different linear IgE-binding motifs. All 28 patients' sera displayed IgE-binding to one specific area (present in Api m 10-isoforms 1 and 2 and putative splice variants 3, 4, 6), indicating a major IgE-epitope. IgE-inhibition provided evidence that the major epitope makes up less than 50% of the total IgE-binding capacity, suggesting that additional (most likely conformational) IgE-epitopes play an important role in Api m 10-sensitization. Api m 10-specific murine IgG and human IgE both predominantly bind to seven different AA-motifs, of which six are identical between both species. Api m 10-isoforms 1 and 2 displayed secondary structural elements and appeared to be aggregated.

The structural, clinical and preclinical insights into Api m 10 and its immunodominant epitopes gained in this study provide substantial insights for the future development of active and passive VIT as well as further treatment approaches.