Hypoallergenic Variants of the Major Latex Allergen Hev b 6.01 Retaining Human T Lymphocyte Reactivity

Abstract

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

Recombinant lipid transfer protein Tri a 14: a novel heat and proteolytic resistant tool for the diagnosis of baker's asthma

“…rTri a 14, nTri a 14 and a wheat salt-soluble protein preparation were tested at 0.1-1 |ig/mL for activation of basophils from three whole blood samples of baker asthmatic donors and one atopic non-asthmatic donor based on analysis of up-regulation of surface expression of CD63 on IgE-positive basophils by flow cytometry [25]. The tests were considered positive when the stimulation index (% of activated basophils with allergen/% of activated basophils at baseline conditions) was > 5.…”

Seed-based oral vaccines as allergen-specific immunotherapies

“…Disruption of conformational epitopes (tertiary structure required for specific IgE binding) can be achieved either by expressing allergen molecules as fragments, reconstruction (reassembling) by molecular scuffling or by mutagenesis of conserved cysteine residues involved in disulphide bonds. Thus, deletion of disulphide bonds required for tertiary structure formation from grass pollen allergen Phl p 7, 60 house dust mite allergen Der f 2, 61 and rubber latex allergen Hev b 6.01, 62 resulted in a reduction of between 40% to 100% of IgE reactivity. Reconstruction of timothy glass pollen allergens, Phl p 1, 63 and Phl p 2, 64 reduced their IgE reactivity to more than 85%.…”

Anaphylaxis to Gelofusine® confirmed by in vitro basophil activation test: a case series*

“…Figure 3 Summary of CD63 expression on basophils incubated with Gelofusine diluted 1 : 5 (solid bar) or 1 : 2 (white bar ) in stimulation buffer and neat (grey bar) for patients sensitive to Gelofusine (1-6), healthy controls (7-9) and control patients allergic to neuromuscular blocking drugs (10)(11)(12)(13)(14). The dotted line shows the cut-off of 3.6% as determined by a receiver operating characteristic curve analysis.…”

“…A reliable blood test to predict or confirm allergy to Gelofusine would enable a simpler and safer management strategy for individuals with possible Gelofusine allergy. The flow cytometric basophil activation test (BAT), with detection of surface expression of CD63 (a lysosomal membrane glycoprotein) on activated basophils, has been useful in identifying subjects allergic to neuromuscular blocking drugs [9] and beta-lactams [10] and more recently we have used this assay to confirm hypoallergenic latex allergens [11]. We report a case series where peri-operative anaphylaxis due to Gelofusine was confirmed by skin testing and BAT, suggesting that the BAT is a reliable screening test for Gelofusine allergy in such patients, avoiding the risks associated with skin testing.…”