Tropomyosins in mosquito and house dust mite cross‐react at the humoral and cellular level

Aedes aegypti and Dermatophagoides pteronyssinus contain important allergens including cross‐reactive tropomyosins. However, the functional and clinical relevance of their cross‐reactivity is still debated.

demonstrated. 6 HDM and mosquitoes are evolutionarily related as part of the Phylum Arthropoda. The mosquito species Aedes aegypti is also a source of several allergens [6][7][8][9] and contains Aed a 10 (tropomyosin). 8,9 We previously demonstrated that natural and recombinant A. aegypti tropomyosins cross-react with tropomyosin from HDM. 8 Tropomyosins belong to a two-stranded alpha-helical coiled coil protein family, which can induce allergic sensitization by ingestion (seafood), inhalation (mites, cockroaches) or parasite infection. 10,11 In developed countries, sensitization to tropomyosin is low. 12,13 However, in some areas in Japan and Africa, the sensitization rate against Der f 10 and Der p 10 is higher than 80% and 55%, respectively. 14,15 In Colombia, sensitization to tropomyosins from mite and Ascaris ranges from 34.6% to 47.7%. 11 The molecular aspects of allergenic cross-reactivity among the different tropomyosins have been studied. Ayuso et al 16,17 described eight peptides which correspond to five putative cross-reactive IgE-binding epitopes sharing 56%-98% of homology with tropomyosins from shrimp, lobster, HDM and cockroach. These regions coincided with five IgE-binding mimotopes described by Leung et al 18 T-cell epitopes from shrimp and cockroach tropomyosins restricted to multiple MHC class II alleles have also been described. 19,20 Aedes aegypti contains two variants of tropomyosin, Aed a 10.0101 and Aed a 10.0201, 8 which slightly differ in their primary structure. Aed a 10.0101 has higher homology to other allergenic tropomyosins and higher conservation of the putative IgE-binding epitopes described by Ayuso et al. 16,17 Based on ELISA competition experiments and sequence analyses, we previously suggested that Aed a 10.0101 contains a higher number of IgE-binding epitopes, is more allergenic and may be more cross-reactive than Aed a 10.0201. 8 To confirm this hypothesis, we analysed the molecular, humoral and cellular cross-reactivity of recombinant (r) tropomyosins from A. aegypti (Aed a 10.0101 and Aed a 10.0201) and from HDM (Der p 10). The humoral cross-reactivity was addressed by immunoassays using sera from Der p 10-sensitized HDM-allergic patients and mice immunized with either Aed a 10.01 or Aed a 10.02. The capacity to induce mediator release was tested by basophil activation tests (BAT). The cellular cross-reactivity and identification of T cell-activating regions were addressed using splenocytes from mice immunized with mosquito-derived tropomyosins.

| Human sera
In total, sera from 15 patients (9 female, 6 male, median ages of 25 years,
Measurements were taken between 250 and 190 nm, with 0.5 nm resolution at a scanning speed of 50 nm/min. Three independent measurements were recorded and averaged for each spectral point.
The final spectra were baseline corrected by subtracting the corresponding buffer spectrum. Results were expressed as the mean residue ellipticity (θ) at a given wavelength.

| Murine antibody responses
ELISA plates were coated and saturated as described above. Sera were diluted 1:500 and incubated overnight. Bound antibodies were detected with rat anti-mouse IgG1 antibodies (BD Pharmingen), followed by a HRP-conjugated goat anti-rat IgG (GE Healthcare, Vienna, Austria). ABTS was added, and the reaction was measured at 405 nm.

| Tropomyosins of A. aegypti and HDM have a similar secondary structure
Amino acid sequence alignment revealed that rDer p 10 shares   between Aed a 10.01, Aed a 10.2 and Der p 10. Therefore, we conclude that T cells specific for epitopes located in these regions may cross-react with tropomyosins from various allergen sources.
Thereby, exposure to Aed a 10 may stimulate the T-cell response initially mounted against allergenic tropomyosins from mosquitoes or any other arthropod species as previously suggested for allergens from HDM and Ascaris. 24 In summary, we have demonstrated that tropomyosins from