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.

The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens with a molecular mass of ~25–30 kDa. Group 1 allergens are recognized by >95% of grass pollen allergic patients. We investigated the IgE recognition of Phl p 1 using allergen-specific IgE-derived single-chain variable Ab fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollen–allergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1–derived peptides, the binding sites of two ScFvs were mapped to the N terminus of the allergen. In surface plasmon resonance experiments they showed comparable high-affinity binding to Phl p 1 as a complete human IgE-derived Ab recognizing the allergens’ C terminus. In a set of surface plasmon resonance experiments simultaneous allergen recognition of all three binders was demonstrated. Even in the presence of the three binders, allergic patients’ polyclonal IgE reacted with Phl p 1, indicating high-density IgE recognition of the Phl p 1 allergen. Our results show that multiple IgE Abs can bind with high density to Phl p 1, which may explain the high allergenic activity and sensitizing capacity of this allergen.

One of the most important classes of reagents for clinical diagnosis is antibodies, either in polyclonal preparations, as monoclonal antibodies (MAbs), or as customized reagents prepared by genetic engineering. The enormous range of antibodies produced by hybridoma and recombinant technologies, together with the requirements of clinical diagnosis for sensitivity and selectivity, make heavy demands on the processes of selecting and characterizing suitable antibody reagents.

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