Mutational analysis of immunodominant epitopes of caprine β‐casein recognized by IgE antibodies from patients allergic to goat’s milk and tolerant to cow’s milk

Background Several cases of allergy to goat’s milk (GM) without allergy to cow’s milk (CM) have been reported. GM-allergy has also been reported in CM-allergic children successfully treated with oral immunotherapy. We previously demonstrated that IgE antibodies from GM-allergic/CM-tolerant patients recognize the caprine b-casein (bcap) without cross-reacting with the bovine b-casein (bbov) despite a high sequence identity (91%). We aimed in the present work to identify the critical amino acids in the non-crossreactive IgE-binding epitopes of bcap. Methods Using site-directed mutagenesis, recombinant bcap was modified by performing residue substitutions with the corresponding amino acids found in bbov. The IgEbinding capacity of the different modified bcap was then evaluated with sera from 9 GM-allergic/CM-tolerant patients and 9 CM-allergic patients. The specificity of murine monoclonal antibodies (mAb) raised against caprine caseins was also analyzed in order to further characterize non-cross-reactive epitopes. The allergenic activity of recombinant bcap was finally assessed by degranulation tests of RBL cells passively sensitized with human IgE antibodies. Results The substitutions A55T/T63P/L75P in the N-terminal part and P148H/S152P in the C-terminal part of bcap induced the greatest decrease of IgE-reactivity of GMallergic/CM-tolerant patients toward the caprine allergen. The threonine 63 was found to be particularly critical, as confirmed by the specificity of mAb SCB1D, whose ability to bind bcap was abolished by the substitution T63P. The recombinant bcap containing the five substitutions was unable to induce the degranulation of RBL cells passively sensitized with IgE from GM-allergic/CM-tolerant patients but was still fully allergenic when testing sera from CM-allergic patients. Conclusion