Does clinical outcome of birch pollen immunotherapy relate to induction of blocking antibodies preventing IgE from allergen binding? A pilot study monitoring responses during first year of AIT

Abstract

Background: The clinical benefit of allergen-specific immunotherapy (AIT) involves induction of blocking antibodies. It is not clear if these antibodies function via steric hindrance alone or a combination of levels, avidities, and epitope specificities, and clinical outcome cannot be predicted. We aim to in-depth characterize serum antibody profiles during birch pollen AIT, investigate therapy-induced antibodies for their capacity to block IgE binding to Bet v 1 and correlate data with clinical outcomes.

Methods: Immune responses of five birch pollen allergic patients were monitored during the first year of AIT by nasal provocation tests (NPTs), ImmunoCAP, immunoblots, direct and avidity enzyme-linked immunosorbent assays, mediator release assays, facilitated antigen binding (FAB) assays, and inhibition mediator release assays.

Results: There was no correlation between NPT results and therapy-induced changes in levels (IgE, IgG, IgA, IgM), avidities, or mediator release potency of Bet v 1-specific antibodies. In FAB assays, blocking antibodies initiated upon AIT were shown to prevent formation of Bet v 1-IgE complexes of an indicator serum pool and significantly correlated with clinical readout. Inhibition mediator release assays using patient-specific IgE for passive sensitization revealed therapy-induced blocking capacities with very good correlation to NPT results. Notably, this assay was the only one to detect a non-responder during treatment in this pilot study.

Conclusions: Clinical outcome of AIT depends on induction of blocking antibodies able to prevent the patient's own IgE from allergen binding. Monitoring of clinical efficacy seems to be best achieved using the inhibition mediator release assay, as development of relevant blocking antibodies can be verified in a patient-tailored manner.

Keywords: Allergens and epitopes; Bet v 1; Birch pollen; Blocking capacity; FAB assay; IgE; Immunotherapy; Inhibition mediator release assay.

Fig. 1

AIT timeline and study design. Subcutaneous immunotherapy with birch pollen extract was performed with 11–13 injections at intervals of 1–2 weeks during the updosing phase. After reaching the maintenance dose, injections were further administered every 4–8 weeks until 52 weeks. Serum samples were obtained before treatment (T0), 2 weeks after reaching maintenance dose (T1), and 1 year after starting treatment (T2). At the same time points, NPT were performed and RTSS were noted

Fig. 2

Reactivity of different antibody subclasses against birch pollen proteins and therapy-related changes. Immunoblot experiments with birch pollen extract and sera of birch pollen AIT patients (P1–5) collected at three time points of immunotherapy (T0, T1, and T2). Bet v 1 is framed in black; NA, non-allergic serum; NBA, non-birch allergic serum; BC, buffer control

Fig. 3

Bet v 1-specific serum antibody titer during AIT and in control sera analyzed by ELISA. a Therapy-related changes in antibody subclass titer. Statistics were performed with Friedman and Dunn’s post test. *p < 0.05. b Correlation of antibody titer with RTSS. Serum samples were obtained at three different time points (T0, open; T1, semi-filled; T2, filled symbols)

Fig. 4

Bet v 1-specific β-hexosaminidase release during AIT analyzed by mediator release assays. a Therapy-related changes in mediator release triggered by 10 ng/ml Bet v 1. Statistics were performed with Friedman and Dunn’s post test. b Correlation of mediator release with RTSS. Serum samples were obtained at three different time points (T0, open; T1, semi-filled; T2, filled symbols)

Fig. 5

Bet v 1-specific serum antibody avidities during AIT and in control sera analyzed by ELISA. a Therapy-related changes in antibody subclass avidity indices. Statistics were performed with Friedman and Dunn’s post test. b Correlation of antibody avidity indices with RTSS. Serum samples were obtained at three different time points (T0, open; T1, semi-filled; T2, filled symbols)

Fig. 6

Graphical illustration of assays used for measuring the AIT-induced capacity of antibodies to prevent IgE-Bet v 1 binding. FAB (a) and inhibition mediator release assays with T0 serum of each patient (b) or indicator serum pool (c) used as reference for therapy-induced antibodies to inhibit IgE-allergen binding

Fig. 7

Inhibition of IgE-Bet v 1 binding during AIT and in control sera. Inhibition assays and correlation of complex destruction with RTSS are given for FAB (a, b), inhibition mediator release assays with T0 serum of each patient (c, d) and indicator serum pool (e, f) used as reference for therapy-induced antibodies to inhibit IgE-allergen binding. Statistics were performed with Friedman test and Dunn’s post test. *p < 0.05, ***p < 0.001. Serum of AIT patients (P1–P5) were obtained at three different time points (T0, open; T1, semi-filled; T2, filled symbols)