In Search of a Vaccine for Mouse Allergy: Significant Reduction of Mus m 1 Allergenicity by Structure-Guided Single-Point Mutations

Abstract

Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. Conclusions: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

Contributions and Future Directions for Structural Biology in the Study of Allergens

“…There are a few nice cases where the structure of the allergens was used to suggest specific mutations to ablate antibody binding by destabilizing the proteins. For example, the structure of the allergen Mus m 1 was used to design a destabilized allergen with reduced IgE binding while retaining T-cell reactivity [46]. In silico screening of point mutations of Bet v 1 and Phl p 5 demonstrated similar results and in addition immunization with the hypoallergenic mutants produced antibodies that blocked human IgE epitopes [47].…”

New Vaccines for Mammalian Allergy Using Molecular Approaches

“…This mutation induced a spatial rearrangement of aromatic side chains and a lower allergenic activity although the T cell reactivity was preserved (27). …”

Food Allergy

“…Protocols for induction of oral tolerance have been described with unmodified allergens, however, the use of natural antigens have a greater chance of eliciting allergic reactions that halt the progression of immunotherapy [81][82][83]. Modified allergens (allergoids) have been used for desensitization protocols to minimize allergic reactions produced by natural antigens [84,85]. Proteins with equivalent epitopes, but less allergenic, can be obtained by gene recombination technology, but this is an expensive methodology [86].…”