Rational Design, Structure–Activity Relationship, and Immunogenicity of Hypoallergenic Pru p 3 Variants

Abstract

Scope Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy. Methods and results Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild‐type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long‐term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE‐binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross‐inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate‐based adjuvant formulation in a mouse model. Conclusions An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.

Dynamics Rationalize Proteolytic Susceptibility of the Major Birch Pollen Allergen Bet v 1

“…Still, the determinants of what makes a protein an allergen are largely unknown (Scheurer et al, 2015;Verhoeckx et al, 2019). It has been demonstrated repeatedly that proteins with substantial similarity in sequence and structure are able to trigger an entirely different immune response (Mitropoulou et al, 2018;Eichhorn et al, 2019;Seutter von Loetzen et al, 2019;Tscheppe et al, 2020). Thus, the allergenic potential of a protein often cannot be rationalized, nor predicted, based on sequence or structural similarity to known allergens (Lu et al, 2018).…”

Critical structural elements for the antigenicity of wheat allergen LTP1 (Tri a 14) revealed by site-directed mutagenesis

“…Indeed, in Par j 1 (LTP from Parietaria judaica pollen) 48 , disruption of Cys14-Cys29 and Cys30-Cys75 bridging caused the loss of speci c IgE-binding and of triggering activity for most patients, whereas a mutant lacking the other bridges retained its activity. The importance of the cysteine doublet in preserving LTP1 structure and allergenicity was also described for Pru p 3 33 . Apart from these two bridges, holding α-helices H1, H2 and H4 together might be an important structural element of LTP1 antigenicity.…”

“…Identi cation of epitopes on plant LTPs has focused on understanding the molecular mechanisms of sensitization to these pan-allergens frequently involved in cross-reactions. Findings would be useful in the context of allergen immunotherapy that aims at developing safe and effective treatment of food allergies using peptides, variants or modi ed proteins [29][30][31][32][33] . We previously studied epitope mapping using immobilized synthetic peptides covering amino acid sequences of wheat LTP1 (Tri a 14) and sera from children allergic to wheat in foods.…”

pH-Induced Local Unfolding of the Phl p 6 Pollen Allergen From cpH-MD

“…Moreover, it was reported repeatedly that slightest distinctions in sequence and/or structure suffice to shift the immune reaction from an allergic to a protective one (Scheurer et al, 2015 ; Verhoeckx et al, 2019 ). Moreover, it is known that high similarity in sequence and/or structure to an allergen protein does not necessitate a similar immune response (Mitropoulou et al, 2018 ; Eichhorn et al, 2019 ; Seutter Von Loetzen et al, 2019 ; Tscheppe et al, 2020 ).…”