Background and objective: Goat's milk (GM) allergy associated with tolerance to cow's milk (CM) has been reported in patients without history of CM allergy and in CM-allergic children successfully treated with oral immunotherapy. The IgE antibodies from GM-allergic/CM-tolerant patients recognize caprine -casein (cap) without cross-reacting with bovine -casein (bov) despite a sequence identity of 91%. In this study, we investigated the non-cross-reactive IgE-binding epitopes of cap. Methods: Recombinant cap was genetically modified by substituting caprine domains with the bovine counterparts and by performing site-directed mutagenesis. We then evaluated the recognition of modified cap by IgE antibodies from 11 GM-allergic/CM-tolerant patients and 11 CM-allergic patients or by monoclonal antibodies (mAb) raised against caprine caseins. The allergenic potency of modified cap was finally assessed by degranulation tests of humanized rat basophil leukaemia (RBL)-SX38 cells. ResultsNon-cross-reactive epitopes of cap were found in domains 44-88 and 130-178. The substitutions A55T/T63P/L75P and P148H/S152P induced the greatest decrease in IgE reactivity of GM-allergic/CM-tolerant patients towards cap. The pivotal role of threonine 63 was particularly revealed as its substitution also impaired the recognition of cap by specific mAb, which could discriminate between cap and bov. The modified cap containing the five substitutions was then unable to trigger the degranulation of RBL-SX38 cells passively sensitized with IgE antibodies from GM-allergic/CM-tolerant patients. Conclusions: Although IgE-binding epitopes are spread all over cap, a non-cross-linking version of cap was generated with only five amino acid substitutions and could thus provide new insight for the design of hypoallergenic variants.