Abstract
BackgroundRecombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild‐type allergens. However, so far no treatment‐induced IgG antibodies have been characterized.ObjectiveTo clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject.MethodsA phage‐displayed combinatorial single‐chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1‐reactive antibody fragments. ScFvs were tested for specificity and cross‐reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross‐reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients’ IgE binding to ELISA plate‐bound allergens and allergen‐induced basophil activation was assessed.ResultsA combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3‐1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients’ polyclonal IgE binding to Bet v 1, and partially suppressed allergen‐induced basophil activation.ConclusionImmunization with unfolded hypoallergenic allergen derivatives induces high‐affinity antibodies even in nonallergic subjects which recognize the folded wild‐type allergens and inhibit polyclonal IgE binding of allergic patients.
Highlights
Allergen-specific immunotherapy (AIT) is an effective, disease-modifying, and long-lasting treatment for IgE-associated allergy.[1-3]
The H3-1 heavy chain (H3-1_HV) showed 99.65% nucleotide sequence identity with the closest related germline ancestor V gene IGHV1-69*09 (Figure 2A) and differed only in one nucleotide located in CDR2 and two in CDR3 resulting in two amino acid exchanges (Data S1, Table S2)
Our study is the first to report the molecular characterization of an allergen-specific IgG antibody induced by immunization with recombinant hypoallergenic allergen derivatives in a nonallergic subject
Summary
Allergen-specific immunotherapy (AIT) is an effective, disease-modifying, and long-lasting treatment for IgE-associated allergy.[1-3]. Several clinical studies performed with such recombinant allergen derivatives and synthetic allergen fragments lacking the folding of natural allergens indicate that the treatment is clinically effective and is associated with the induction of allergen-specific IgG antibodies which compete with allergic patients[0] IgE for binding to the natural, native allergens.[13-16]. Immunization with recombinant, unfolded fragments of the major birch pollen allergen Bet v 1 raised IgG antibodies against Bet v 1 peptides,[17] but serum from the immunized patients inhibited binding of allergic patients IgE antibodies against conformational epitopes. To investigate IgG antibodies induced with recombinant unfolded allergen derivatives in detail, we immunized nonallergic subjects with recombinant Bet v 1 fragments and constructed a combinatorial single-chain fragment library from an immunized subject who developed IgG antibodies recognizing the native allergen. Our results demonstrated that IgG antibodies induced with recombinant, unfolded allergen derivatives simultaneously recognize peptide epitopes as well as epitopes on the folded native allergen and can act as blocking antibodies
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