Designing a Hypoallergenic Mutant of Cra a 2 with T-Cell Reactivity

Crossostrea angulata arginine kinase (Cra a 2) is a major allergen responsible for oyster allergy, emphasizing the need to develop its hypoallergenic mutant for oral desensitization. In this study, a mutant Cra a 2 (mCra a 2) was generated by simultaneously substituting the four key amino acids within the conformational B-cell epitopes. These site-directed mutations altered the structure and surface electrostatic potential, leading to reduction in immunoglobulin (Ig)G-/IgE-binding capacity and attenuated basophil activation, thereby confirming the diminished allergenic potential of mCra a 2. For T-cell reactivity analysis, two T-cell epitopes were identified and found to remain preserved in mCra a 2, thereby preserving its ability to activate T-cell. Notably, splenocytes from wild-type Cra a 2-immunized mice stimulation with mCra a 2 up-regulate IL-10 secretion. Collectively, mCra a 2 not only exhibits attenuated immunoreactivity but also retains robust T-cell reactivity, suggesting its potential utility in oral desensitization of oyster allergy.