Goat's milk allergy without cow's milk allergy: suppression of non-cross-reactive epitopes on caprine ß-casein.

ABSTRACT

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 ß-casein (ßcap) without cross-reacting with bovine ß-casein (ßbov) despite a sequence identity of 91%. In this study, we investigated the non-cross-reactive IgE-binding epitopes of ßcap. METHODS: Recombinant ßcap was genetically modified by substituting caprine domains with the bovine counterparts and by performing site-directed mutagenesis. We then evaluated the recognition of modified ßcap 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 ßcap was finally assessed by degranulation tests of humanized rat basophil leukaemia (RBL)-SX38 cells. RESULTS: Non-cross-reactive epitopes of ßcap 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 ßcap. The pivotal role of threonine 63 was particularly revealed as its substitution also impaired the recognition of ßcap by specific mAb, which could discriminate between ßcap and ßbov. The modified ßcap 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 ßcap, a non-cross-linking version of ßcap was generated with only five amino acid substitutions and could thus provide new insight for the design of hypoallergenic variants.