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In addition, a fold variant of FPH termed FPH4 was designed by in silico mutagenesis.
Thus, we tested the low IgE-binding, highly immunogenic fold variant BM4 in a Bet v 1 mouse model.
This was not expected, given that the Bet v 1 fold variant BM4 was recently shown to display increased resistance toward proteolytic digestion (15).
As previously published, a fold variant of Bet v 1.0101 (termed Bet v 1 thereafter), BM4, is suggested to improve efficacy of SIT.
For either population, both variants showed reduced IgE-binding capacities; however, this effect was most striking for the fold variant FPH4.
To investigate the effects of BM4 as novel therapeutic, we compared the impact of recombinant Bet v 1 and, its fold variant BM4 in a therapeutic mouse model.
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The proteins were produced by site-directed mutagenesis as fold variants of their parental allergens.
Notably, both in vivo models revealed reduced immunogenicity of the hypoallergenic fold variants.
Using this method, we found that the fold variants showed a higher susceptibility toward proteolysis, a fact that could explain their low immunogenic potential.
This lead to the production of the cysteine variants (CVs) Mal d 1 C107S and Cor a 1 C4,82,160S as well as the cysteine fold variants (CFVs) Mal d 1 C107S, I113K and Cor a 1 C4,82,160S, L115K (Fig. 1A).
Thus, the cysteins were replaced by serines to mimic the behavior of monomeric Bet v 1. Cysteine and cysteine fold variants were generated, which did not form aggregates any longer.
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