We assessed the effect of multiple-mutations within one IgE-binding area on allergenicity of Der f 2. The triple-mutant of Der f 2, P34/95/99A, exhibited the most significant reduction of allergenicity and circular dichroism analysis showed that the global structure of Der f 2 was maintained in P34/95/99A. These results indicate that such a strategy is effective when designing allergen-vaccines, which achieve less allergenicity for a broad population of patients without disrupting the global structure. Structurally, Der f 2 is a member of the MD-2 related lipid-recognition proteins. The sites for the triple-mutation located on the characteristically charged entrance of a cavity and corresponded to the regions critical to ligand-binding in the Niemann-Pick type 2 disease protein and MD-2.