Monitoring the epitope recognition profiles of IgE, IgG 1 , and IgG 4 during birch pollen immunotherapy
Authors: Barbara Gepp, Nina Lengger, Christian Möbs, Wolfgang Pfützner, Christian Radauer, Barbara Bohle, Heimo Breiteneder
Journal: Journal of Allergy and Clinical Immunology (2015)

It is well established that allergen-specific IgG antibodies induced during allergen-specific immunotherapy (AIT) can block the IgE-mediated cascade of allergic inflammation.1Flicker S. Valenta R. Renaissance of the blocking antibody concept in type I allergy.Int Arch Allergy Immunol. 2003; 132: 13-24Crossref PubMed Scopus (139) Google Scholar Although this role of IgG has been intensively studied, little is known about the epitope diversities of immunoglobulin subclasses induced during AIT. Thus far, analyses of IgG4-binding patterns to major cow's milk2Savilahti E.M. Kuitunen M. Valori M. Rantanen V. Bardina L. Gimenez G. et al.Use of IgE and IgG4 epitope binding to predict the outcome of oral immunotherapy in cow's milk allergy.Pediatr Allergy Immunol. 2014; 25: 227-235Crossref PubMed Scopus (53) Google Scholar and peanut3Vickery B.P. Lin J. Kulis M. Fu Z. Steele P.H. Jones S.M. et al.Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens.J Allergy Clin Immunol. 2013; 131: 128-134Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar allergens during oral AIT revealed discrepant results. The overall diversity of IgG4 specific for cow's milk allergens changed little,2Savilahti E.M. Kuitunen M. Valori M. Rantanen V. Bardina L. Gimenez G. et al.Use of IgE and IgG4 epitope binding to predict the outcome of oral immunotherapy in cow's milk allergy.Pediatr Allergy Immunol. 2014; 25: 227-235Crossref PubMed Scopus (53) Google Scholar whereas the IgG4 repertoire specific for peanut allergens was broadly expanded in a polyclonal fashion, including de novo–generated specificities different from IgE.3Vickery B.P. Lin J. Kulis M. Fu Z. Steele P.H. Jones S.M. et al.Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens.J Allergy Clin Immunol. 2013; 131: 128-134Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar Binding of IgE to Bet v 1, the major birch pollen allergen, is dependent on the protein's native conformation.4Vrtala S. Hirtenlehner K. Vangelista L. Pastore A. Eichler H.G. Sperr W.R. et al.Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy.J Clin Invest. 1997; 99: 1673-1681Crossref PubMed Scopus (206) Google Scholar We have previously assessed the diversity of IgG4 antibodies specific for Bet v 1 during subcutaneous AIT with birch pollen using competitive immunoscreening of phage-displayed peptides and found that the Bet v 1–specific IgG4 repertoire did not broadly expand in most patients.5Subbarayal B. Schiller D. Möbs C. Pfützner W. Jahn-Schmid B. Gepp B. et al.The diversity of Bet v. 1-specific IgG4 antibodies remains mostly constant during the course of birch pollen immunotherapy.J Allergy Clin Immunol. 2015; ([E-pub ahead of print]. doi: http://dx.doi.org/10.1016/j.jaci.2015.05.025)PubMed Google Scholar However, competitive immunoscreening is laborious and can be performed only with a limited number of serum samples. Therefore our previous work was restricted to the analysis of IgG4 diversity at 2 time points (before and after 3 years) of AIT by using sera of 5 patients. Here, we applied a chimera-based approach to monitor development of the Bet v 1–specific IgE, IgG1, and IgG4 repertoires in narrow time intervals during 3 years of AIT in more subjects.6Mobs C. Slotosch C. Loffler H. Jakob T. Hertl M. Pfutzner W. Birch pollen immunotherapy leads to differential induction of regulatory T cells and delayed helper T cell immune deviation.J Immunol. 2010; 184: 2194-2203Crossref PubMed Scopus (108) Google Scholar Four chimeras were generated by grafting 4 different nonoverlapping, contiguous, Bet v 1–derived surface areas onto the Bet v 1 homolog from celeriac, Api g 1, by replacing Api g 1–specific surface residues by corresponding residues from Bet v 1 (see Fig E1 in this article's Online Repository at www.jacionline.org).7Gepp B. Lengger N. Bublin M. Hemmer W. Breiteneder H. Radauer C. Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy.J Allergy Clin Immunol. 2014; 134: 188-194Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Api-Bet-1 contains the P-loop, Api-Bet-2 contains the region opposite the P-loop, Api-Bet-3 contains the C-terminus and surrounding residues, and Api-Bet-4 contains the C-terminal α-helix of Bet v 1. Sera from 11 patients with birch pollen allergy with improved symptom and medication scores were collected before (time point 0) and after 1, 3, 6, 12, 18, 24, 30, and 36 months of AIT.6Mobs C. Slotosch C. Loffler H. Jakob T. Hertl M. Pfutzner W. Birch pollen immunotherapy leads to differential induction of regulatory T cells and delayed helper T cell immune deviation.J Immunol. 2010; 184: 2194-2203Crossref PubMed Scopus (108) Google Scholar Patients received weekly doses of birch pollen extract (ALK-depot SQ 108; ALK-Abelló, Hørsholm, Denmark) subcutaneously until a maintenance dose of 100,000 standard quality units per injection was reached, followed by monthly maintenance injections for 3 years. Antibody binding to Bet v 1, the 4 Bet v 1–specific areas on the chimeras, and Api g 1 (scaffold protein control) was assessed by means of ELISA. Sera were not available from all time points from 3 subjects (p1, p5, p8). Proteins (2 μg/mL) were coated onto 96-well microtiter plates (MaxiSorp; Nalge Nunc International, Roskilde, Denmark) in carbonate buffer (50 mmol/L, pH 9.6). Nonspecific binding sites were saturated for 2 hours at room temperature with 1% human serum albumin in Tris-buffered saline containing 0.5% Tween-20 (TBST). Sera were diluted 1:20 (IgE), 1:50 (IgG1), or 1:200 (IgG4) in TBST with 0.5% human serum albumin and applied in duplicates overnight at 4°C. Bound antibodies were detected with alkaline phosphatase–conjugated mouse anti-human IgE (BD PharMingen, Heidelberg, Germany), IgG1 (Acris Antibodies, Herford, Germany), or IgG4 (BD PharMingen) mAbs. OD was measured after color development with Sigma FAST p-nitrophenyl phosphate tablets (Sigma-Aldrich, St Louis, Mo) at 405 nm. Sera from 8 nonallergic subjects served as negative controls. OD values for these controls were not significantly different from the buffer controls when IgE, IgG1, or IgG4 binding to the proteins was assessed. For all 11 immunotherapy sera, OD values exceeding the mean value of the 8 nonallergic subjects by more than 5 SDs were considered positive. To assess specific antibody binding to the grafted region on each chimera, the OD value measured for the scaffold protein Api g 1 was subtracted from the chimera-specific OD value and considered positive when the difference exceeded 5 times the SD of the negative control values. In the majority of patients, Bet v 1–specific IgE levels increased during the early phase of treatment, followed by a gradual decrease (Fig 1). Seven (64%) of 11 patients displayed lower IgE levels after 36 months of AIT compared with those before therapy (Fig 1). All patients had Bet v 1–specific IgG4, and 7 (64%) of 11 patients had Bet v 1–specific IgG1 (Fig 1). In these subjects Bet v 1–specific IgG1 antibody levels increased earlier than IgG4 antibody levels (median duration required to reach half-maximum concentrations of Bet v 1–specific IgG1 and IgG4: 4.5 and 19.5 months; P = .016, Wilcoxon signed-rank test). Accordingly, chimera-specific IgG1 appeared earlier than IgG4 (Fig 2).Fig 2IgE, IgG1, and IgG4 binding to Api g 1 and the 4 Bet v 1–specific areas on chimeras of 11 patients during AIT with birch pollen. OD values for Api g 1 (scaffold protein) were subtracted from chimera-specific OD values.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The patterns of IgE recognition of the 4 Bet v 1–specific areas on the chimeras and Api g 1 did not change over time in most patients, indicating that the overall IgE epitope diversity during AIT remained constant (Fig 2). Similarly, once induced, chimera recognition was constant in 4 (57%) of 7 patients (p3, p5, p7, and p10) for IgG1 and 8 (73%) of 11 patients (p1-p5, p7, p8, and p11) for IgG4, albeit with quantities varying over time (Fig 2). These findings match our previous results achieved by means of competitive immunoscreening of phage-displayed peptides, showing that the early established IgG4 repertoire did not change with prolonged AIT.5Subbarayal B. Schiller D. Möbs C. Pfützner W. Jahn-Schmid B. Gepp B. et al.The diversity of Bet v. 1-specific IgG4 antibodies remains mostly constant during the course of birch pollen immunotherapy.J Allergy Clin Immunol. 2015; ([E-pub ahead of print]. doi: http://dx.doi.org/10.1016/j.jaci.2015.05.025)PubMed Google Scholar IgE binding to all 4 chimeras was observed in 7 (64%) of 11 patients (Fig 2). In contrast, only 2 (p3 and p10) and 3 (p2, p4, and p7) patients showed IgG1 or IgG4 binding to all chimeras, respectively. We conclude that among the immunoglobulin classes investigated, IgE showed the highest epitope diversity. Therefore, most likely, not all IgE-binding epitopes could be blocked by AIT-induced IgG1 or IgG4 because of direct epitope competition. It was shown that IgA, IgG2, and IgG3 also possess blocking activity,8Dodev T.S. Bowen H. Shamji M.H. Bax H.J. Beavil A.J. McDonnell J.M. et al.Inhibition of allergen-dependent IgE activity by antibodies of the same specificity but different class.Allergy. 2015; 70: 720-724Crossref PubMed Scopus (47) Google Scholar which could contribute to the reported amelioration of allergic symptoms. However, IgG2 and IgG3 were not induced during AIT,5Subbarayal B. Schiller D. Möbs C. Pfützner W. Jahn-Schmid B. Gepp B. et al.The diversity of Bet v. 1-specific IgG4 antibodies remains mostly constant during the course of birch pollen immunotherapy.J Allergy Clin Immunol. 2015; ([E-pub ahead of print]. doi: http://dx.doi.org/10.1016/j.jaci.2015.05.025)PubMed Google Scholar and IgA induction was low.6Mobs C. Slotosch C. Loffler H. Jakob T. Hertl M. Pfutzner W. Birch pollen immunotherapy leads to differential induction of regulatory T cells and delayed helper T cell immune deviation.J Immunol. 2010; 184: 2194-2203Crossref PubMed Scopus (108) Google Scholar Steric inhibition by binding of IgG to sites adjacent to IgE epitopes on the grafted regions could also contribute to IgE blocking.1Flicker S. Valenta R. Renaissance of the blocking antibody concept in type I allergy.Int Arch Allergy Immunol. 2003; 132: 13-24Crossref PubMed Scopus (139) Google Scholar In addition, mechanisms not depending on epitope-matched blocking IgG antibodies might contribute to the success of immunotherapy by mixed immune complexes containing IgE and IgG, such as inhibition of IgE-facilitated antigen presentation, as shown in our previous study,5Subbarayal B. Schiller D. Möbs C. Pfützner W. Jahn-Schmid B. Gepp B. et al.The diversity of Bet v. 1-specific IgG4 antibodies remains mostly constant during the course of birch pollen immunotherapy.J Allergy Clin Immunol. 2015; ([E-pub ahead of print]. doi: http://dx.doi.org/10.1016/j.jaci.2015.05.025)PubMed Google Scholar and inhibition of mast cell degranulation. Furthermore, our data revealed that IgE, IgG1, and IgG4 recognized different epitope profiles (Fig 2). For example, patient 1 showed IgE binding exclusively to Api-Bet-2 and Api-Bet-3 but had high amounts of IgG4 directed against Api-Bet-1. Patient 3 had IgE and IgG1 binding mainly to Api-Bet-1, Api-Bet-2, and Api-Bet-3 but developed solely Api-Bet-3–specific IgG4. For patient 7, IgE and IgG4 binding to all 4 chimeras was observed, whereas Api-Bet-2–specific IgG1 was not detected. In summary, this is the first study that longitudinally monitored IgE, IgG1, and IgG4 repertoires in narrow intervals during 3 years of subcutaneous AIT. The grafted Bet v 1–specific areas on our chimeras are approximately 2000 Å2 offering potential space for 2 or 3 epitopes7Gepp B. Lengger N. Bublin M. Hemmer W. Breiteneder H. Radauer C. Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy.J Allergy Clin Immunol. 2014; 134: 188-194Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and allow an approximation of the epitope diversities of different allergen-specific immunoglobulin subclasses. Our data provide evidence that the allergen-specific IgE repertoire and the AIT-induced IgG1 and IgG4 profiles did not expand over time but differed among each other. Our finding that AIT-induced IgG1 antibodies developed earlier than IgG4 antibodies is in agreement with previous reports that IgG1 dominates the early and IgG4 dominates the late IgG response in patients with AIT.9Djurup R. The subclass nature and clinical significance of the IgG antibody response in patients undergoing allergen-specific immunotherapy.Allergy. 1985; 40: 469-486Crossref PubMed Scopus (133) Google Scholar Both IgG1 and IgG4 displayed more restricted epitope diversities than IgE antibodies. Together, our findings contribute to elucidating the complex immune mechanisms that occur during AIT.