rAed a 4: A New 67-kDa <b><i>Aedes aegypti</i></b> Mosquito Salivary Allergen for the Diagnosis of Mosquito Allergy

Abstract

Background: Accurate diagnosis of mosquito allergy has been hampered by the laborious task of obtaining mosquito salivary allergens. We have previously studied 3 recombinant (r) Aedes aegypti mosquito salivary allergens: rAed a 1, rAed a 2 and rAed a 3. Here, we report the expression, purification, identification and evaluation of rAed a 4, a 67-kDa α-glucosidase. Methods: rAed a 4 was expressed using a baculovirus/insect cell system, purified by a combination of anion- and cation-exchange chromatography, and identified by immunoblotting. A. aegypti saliva extract was prepared in our laboratory. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to measure rAed a 4-specific immunoglobulin E (IgE) and IgG antibodies in sera from 13 individuals with a positive mosquito-bite test from a laboratory-reared mosquito. Sera from 18 individuals with a negative bite test served as controls. Results: Purified rAed a 4 bound to the IgE in mosquito-allergic sera, as detected by ELISA and immunoblotting. The binding of rAed a 4 to IgE could be inhibited in a dose-dependent manner by the addition of an A. aegypti extract. Mosquito-allergic individuals had significantly higher mean levels of rAed a 4-specific IgE and IgG than controls. Using the mean of the controls ± 2 SD as a cut-off level, 46% of the 13 allergic individuals had a positive IgE, while none of the controls was positive (p < 0.001). Conclusions: Aed a 4 is a major allergen in mosquito saliva. Its recombinant form has the hydrolase function and can be used for the diagnosis of mosquito allergy.

A salivary factor binds a cuticular protein and modulates biting by inducing morphological changes in the mosquito labrum

“…albopictus saliva, we have analyzed the saliva forcedly expectorated by mature females using the cholinomimetic drug Pilocarpine 27 (Table S1; Figure 1A). Wondering which of the identified proteins might be strictly related to blood feeding, considering that female mosquitoes feed also on floral nectars, [28][29][30] we compared the salivary glands secretome from young (1-day-old) and mature (6-day-old) mosquitoes using 2D gel analyses (Figures 1B and S1; distance-based ANOVA of fuzzy maps: pseudo-F = 1.73, p < 0.05; 999 permutations). Aedes mosquitoes, indeed, usually take blood only few days after emergence: 31 we expected that the amount of proteins linked to blood feeding would have increased during sexual maturation, as already noted in the case of SGS proteins in Aedes aegypti.…”

Novel salivary gland allergens from tropical mosquito species and IgE reactivity in allergic patients

“…The most wellcharacterized A. aegypti mosquito salivary allergens are apyrase (Aed a 1), 26 a D7 family protein (Aed a 2) 27 , a 30 kDa salivary protein (Aed a 3) 28 , and alpha-glucosidase (Aed a 4). 29 In addition, 6 non-salivary allergenic proteins were recently characterized: Aed a5 (sarcoplasmic Ca þ binding protein), Aed a6 (Porin3), Aed a 7, Aed a 8 (HSC-70), Aed a 10 (Tropomyosin), 30 and Aed a 11 (lysosomal aspartic protease). 31 The novel salivary allergens identified in this study from A. albopictus are Aed al 2 (33 kDa), a D7 family protein, and Aed al 3 (30 kDa), which has high sequence similarity to Aed a 3.…”

Allergenome characterization of the mosquito Aedes aegypti

“…Additionally, four proteins with inferred biological function and one without known biological function were also identified. Previous studies had focused on the saliva and salivary glands of A. aegypti , and four allergens have been reported as factors associated with the cutaneous reactions that appear after mosquito bites . In this study, we did not detect these allergens as we used sera from individuals with respiratory allergy.…”