Mutant derivatives of the main respiratory allergen of cow are less allergenic than the intact molecule

Background Allergen immunotherapy offers an alternative for drug treatment in the management of allergic diseases. Because immunotherapy often induces side-effects, less allergenic preparations would be beneficial. Objective The purpose of this study was to examine whether the allergenicit y of a cow-derived lipocalin allergen, Bos d 2, could be diminished by subs tituting or deleting carboxyterminal amino acids including the cysteine whi ch forms a disulphide bond with a cysteine inside the molecule. Methods Four recombinant mutants of Bos d 2 were created by substituting or deleting the four most carboxy-terminal amino acids. The immunological cha racteristics of the mutant preparations were compared with the unmodified r Bos d 2 by Western blotting, ELISA inhibition, skin prick tests, and the pr oliferative responses of allergen-specific T-cell clones. Results In Western blot, one of the two monoclonal antibodies showed reduce d binding to the preparations without the terminal cysteine. In contrast, t he other monoclonal antibody, human IgE and rabbit immune serum bound equal ly well to all the preparations. ELISA inhibition analyses revealed, howeve r, that the preparations without the terminal cysteine bound antibody less efficiently. They were needed 15-38 times more than the unmodified rBos d 2 to cause the same level of inhibition. Surprisingly, one of the mutants wi th the terminal cysteine but a mutated adjacent amino acid turned out to be the weakest in inducing skin reactivity. All the preparations stimulated w ell allergen-specific T-cell clones. Conclusions The results show that the allergenicity of a lipocalin allergen , Bos d 2, can be diminished by modifying the carboxy-terminal end of the m olecule. Modifications in the area which encompasses a disulphide bond impa ired the antibody binding without affecting the T-cell stimulatory capacity . It was also shown that in vivo tests are necessary for determining the al lergenicity of a modified allergen.