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The diversity of Bet v 1–specific IgG 4 antibodies remains mostly constant during the course of birch pollen immunotherapy

2015; Elsevier BV; Volume: 136; Issue: 6 Linguagem: Inglês

10.1016/j.jaci.2015.05.025

ISSN

1097-6825

Autores

Brinda Subbarayal, Dirk Schiller, Christian Möbs, Wolfgang Pfützner, Beatrice Jahn‐Schmid, Barbara Gepp, Heimo Breiteneder, Stefan Vieths, Barbara Bohle,

Tópico(s)

Asthma and respiratory diseases

Resumo

It is well documented that allergen-specific immunotherapy (AIT) induces high titers of allergen-specific IgG antibodies, which may block IgE-mediated effector mechanisms by preventing IgE binding to allergens. Both IgG1 and IgG4 antibodies have been considered as blocking antibodies; however, the functional role of allergen-specific IgG4 antibodies has been most intensely studied so far. Still, only limited data on the epitope specificity and diversity of AIT-induced allergen-specific IgG4 antibodies exist. Recently, the antibody repertoire after a median time period of 41 months of oral AIT has been studied in a high-throughput peptide microarray by using linear peptides of major peanut allergens.1Vickery B.P. Lin J. Kulis M. Fu Z. Steele P.H. Jones S.M. et al.Peanut oral immunotherapy modifies IgE and IgG(4) responses to major peanut allergens.J Allergy Clin Immunol. 2013; 131: 128-134.e3Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar The authors concluded that the peanut-specific IgG4 repertoire broadly expanded in a polyclonal fashion and included novel specificities that recognized epitopes different from IgE antibodies.1Vickery B.P. Lin J. Kulis M. Fu Z. Steele P.H. Jones S.M. et al.Peanut oral immunotherapy modifies IgE and IgG(4) responses to major peanut allergens.J Allergy Clin Immunol. 2013; 131: 128-134.e3Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar In a different type of AIT, namely, subcutaneous treatment with birch pollen extract (BP-AIT) containing 5% to 10% of the sole major allergen Bet v 1, we have reported that most Bet v 1–specific IgG4 antibodies induced after 36 months recognized IgE epitopes and only a minority represented de novo specificities.2Subbarayal B. Schiller D. Mobs C. de Jong N.W. Ebner C. Reider N. et al.Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen.Allergy. 2013; 68: 1377-1386Crossref PubMed Scopus (43) Google Scholar In the present longitudinal study, we sought to investigate whether the diversity of Bet v 1–specific IgG4 antibodies expands, decreases, or remains similar in the course of BP-AIT. Because antibody epitopes on Bet v 1 are conformational,3Hecker J. Diethers A. Schulz D. Sabri A. Plum M. Michel Y. et al.An IgE epitope of Bet v 1 and fagales PR10 proteins as defined by a human monoclonal IgE.Allergy. 2012; 67: 1530-1537PubMed Google Scholar, 4Dall'Antonia F. Gieras A. Devanaboyina S.C. Valenta R. Keller W. Prediction of IgE-binding epitopes by means of allergen surface comparison and correlation to cross-reactivity.J Allergy Clin Immunol. 2011; 128: 872-879.e8Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Holm J. Ferreras M. Ipsen H. Wurtzen P.A. Gajhede M. Larsen J.N. et al.Epitope grafting, re-creating a conformational Bet v 1 antibody epitope on the surface of the homologous apple allergen Mal d 1.J Biol Chem. 2011; 286: 17569-17578Crossref PubMed Scopus (33) Google Scholar we performed competitive immunoscreening of phage-displayed peptides to predict IgG4-binding areas2Subbarayal B. Schiller D. Mobs C. de Jong N.W. Ebner C. Reider N. et al.Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen.Allergy. 2013; 68: 1377-1386Crossref PubMed Scopus (43) Google Scholar, 6Mittag D. Batori V. Neudecker P. Wiche R. Friis E.P. Ballmer-Weber B.K. et al.A novel approach for investigation of specific and cross-reactive IgE epitopes on Bet v 1 and homologous food allergens in individual patients.Mol Immunol. 2006; 43: 268-278Crossref PubMed Scopus (68) Google Scholar using sera collected after 6 and 36 months of BP-AIT from 5 BP-allergic patients with significantly improved respiratory symptoms to natural BP exposure and reduced skin prick test reactivity to BP extract.7Mobs C. Ipsen H. Mayer L. Slotosch C. Petersen A. Wurtzen P.A. et al.Birch pollen immunotherapy results in long-term loss of Bet v 1-specific T(H)2 responses, transient T(R)1 activation, and synthesis of IgE-blocking antibodies.J Allergy Clin Immunol. 2012; 130: 1108-1116.e6Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar Briefly, each serum sample was incubated with antihuman IgG4-coated magnetic beads that were then used to screen a phage-displayed peptide library (Ph.D.-12 Phage Display Peptide Library, New England BioLabs, Beverly, Mass). Bound phages were specifically eluted by competition with Bet v 1. The amino acid (aa) sequences of the phage-displayed 12-mer peptides were determined. This procedure was repeated until at least 4 peptides for each serum sample were obtained (see Table E1 in this article's Online Repository at www.jacionline.org). Each peptide sequence was mapped onto the molecular surface of Bet v 1 (PDB code 1BV1) with the "EpiSearch" algorithm to predict conformational regions.8Negi S.S. Braun W. Automated detection of conformational epitopes using phage display peptide sequences.Bioinform Biol Insights. 2009; 3: 71-81Crossref PubMed Google Scholar In total, 36 and 40 IgG4 epitopes, each comprising 10 to 23 aa, were predicted at month 6 and 36 of BP-AIT, respectively (see Table E1). Next, we assessed the percentage of identical aa in the same IgG4-binding Bet v 1–surface areas predicted at both time points. For patient 1, 44.7% aa were identical; for patient 2, 61.5%; for patient 3, 62.5%; for patient 4, 53.8%; and for patient 5, 36.8%. Fig 1 illustrates the entire surface area predicted to be recognized by IgG4 antibodies on Bet v 1 at each time point and their overlap for each individual. The remarkable overlap of predicted IgG4 epitopes at 6 and 36 months of BP-AIT in 4 of the 5 patients (80%) indicated that the early established IgG4 repertoire did not expand further with prolonged treatment. To complement the in silico data, we assessed the IgE-blocking activity by facilitated antibody-binding assays as described.2Subbarayal B. Schiller D. Mobs C. de Jong N.W. Ebner C. Reider N. et al.Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen.Allergy. 2013; 68: 1377-1386Crossref PubMed Scopus (43) Google Scholar Briefly, each serum sample was preincubated with Bet v 1 and the same indicator serum containing more than 100 kU/L of Bet v 1–specific IgE with a presumed highly diverse IgE repertoire. Thereafter, the activity of each serum to reduce the binding of Bet v 1-IgE complexes to CD23+ B cells was determined. Bet v 1–specific IgG1-4 levels in the sera were assessed by ELISA. None of the patients developed Bet v 1–specific IgG2 and IgG3 antibodies (data not shown), and only patients 1, 3, and 4 developed Bet v 1–specific IgG1 antibodies (see Fig E1 in this article's Online Repository at www.jacionline.org). Bet v 1–specific IgG4 antibody levels were also quantified by ImmunoCAP (Thermo Fisher Scientific, Uppsala, Sweden). Table I shows that the IgE-blocking activity of sera from patient 2 had reached 89% already after 6 months of BP-AIT and was not markedly enhanced after 36 months despite further increase in Bet v 1–specific IgG4 antibody levels. The finding that the IgE-blocking activity did not rise in parallel to the quantity of specific IgG4 antibodies suggested that the sera at both time points contained comparable IgG4 specificities able to block most IgE epitopes recognized by the indicator serum. These data accorded with the large overlap of the predicted IgG4-binding regions at both time points (Fig 1). However, patient 4 displayed very high levels of Bet v 1–specific IgG4 antibodies at month 6 and 3.5-fold lower levels at month 36 (Table I). At this time point, Bet v 1–specific IgG1 antibodies were almost reduced to the level before AIT (see Fig E1). In parallel, the IgE-blocking activity decreased only around 2-fold (Table I). The limited reduction in IgE-blocking activity together with the high overlap of predicted IgG4 epitopes at both time points (Fig 1) suggested a comparable IgG4 repertoire in the early and late phases of BP-AIT also in this patient. In contrast, patient 5 showed a parallel increase in IgE-blocking activity and IgG4 levels but no IgG1 induction during the course of BP-AIT (see Table I and Fig E1). In this individual, the increased IgE-blocking activity may result not only from the enhanced IgG4 concentration but also from a broader IgG4 diversity. The latter is strongly suggested by the occurrence of prominent IgG4-binding areas predicted at month 36 but not at month 6 (Fig 1). We conclude that the Bet v 1–specific IgG4 repertoire of patient 5 expanded in the course of BP-AIT.Table IBet v 1–specific IgG4 levels and IgE-blocking activity at month 6 and 36 of BP-AITPatientIgE-blocking activity (%)∗The IgE-blocking activity of sera was assessed by facilitated antibody-binding assays.IgG4 levels (μg/L)†Bet v 1–specific IgG4 levels were determined by ImmunoCAP.BeforeMonth 6Month 36BeforeMonth 6Month 361198980.000.722.492089920.030.581.123096990.286.3426.84086450.5518.55.295036621.221.6711.8∗ The IgE-blocking activity of sera was assessed by facilitated antibody-binding assays.† Bet v 1–specific IgG4 levels were determined by ImmunoCAP. Open table in a new tab In summary, this study provides first evidence that Bet v 1–specific IgG4 repertoires induced by BP-AIT do not broadly expand in the course of treatment in most of the patients. Still, 1 of 5 individuals (20%) showed an increase in the IgG4 diversity with prolonged therapy. We are aware that these conclusions are based on a limited number of patients, in particular in view of the heterogeneous and individual immune response to allergens. However, competitive immunoscreening is laborious and cannot be performed in a high-throughput set-up. Therefore, we sought an additional approach to support our conclusions, namely, the use of 4 chimeric proteins that had been created by grafting surface areas of Bet v 1 onto the scaffold of Api g 1, the Bet v 1–homologue in celery.9Gepp 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 The IgG4 reactivity to these chimeras was tested by ELISA. So far, 8 of 9 patients (89%) displayed IgG4 reactivity to the same chimera after 6 and 36 months of BP-AIT, whereas 1 of 9 individuals (11%) showed a diverse response at both time points (unpublished observations). Identical reactivity patterns at early and late time points of the therapy indicate that the IgG4 repertoire remained constant for most of the patients during BP-AIT. These preliminary data obtained by a second, entirely different experimental approach to analyze the IgG4 diversity in the course of BP-AIT strongly underline our conclusions drawn from competitive immunoscreening of phage-displayed peptides and facilitated antibody-binding assays. Table E1Phage-displayed peptides and predicted IgG4-binding regions on Bet v 1PatientBP-SIT (mo)Peptide sequenceaa residues of predicted IgG4-binding regions on Bet v 1% Of identical aa at 6 and 36 moQSVRIMTGADSN1. S39, I56, S57, G61, V67, V85, G88, G89, I91, G92, Q132, A135, S136, M139KCSLPECVVARE2. S39, K55, S57, P59, E60, L62, P63, K65, V67, K68, V85, E87, P90, A135, S13616SPGLPLQLGSMK3. S39, K55, S57, P59, G61, L62, P63, K65, K68, G88, G89, P90, G92, Q132, S136, M13944.7SKMAHMERSWEV4. V2, E6, V85, K97, S99, E101, K115, S117, K119, H121, V128, V133, K134, A135, S136, K137, M139SKMAHMERSWEV5. E6, V67, V85, S117, K119, V128, K129, A130, E131, V133, K134, A135, S136, K137, E138, M139, E141VQHNTKYSVVIR6. N4, Y5, T7, T9, I23, K80, Y81, N82, Y83, S99, I102, K103, V105, I113, K115, I116, S117, N118, K119, V133, S136, K137VQHNTKYSVVIR7. V2, N4, Y5, T7, N82, Y83, V85, K97, S99, I102, K115, I116, S117, N118, K119, H121, V128, Q132, V133, K134, S136, K137YLDLPTNLSHRI8. T7, T9, I23, L24, D72, D75, N78, Y81, N82, Y83, S99, I102, I113, I116, S117, N118, S136, L143TLRVPPNPNMNV9. T7, T9, L62, P63, V67, V85, P90, N118, V133, M139, T142, L143, R145GHVTESMTKRSD10. G1, V2, V67, V85, E87, G88, G89, G92, D93, T94, E96, K97, S99, K119, H121, T122, K123, G124, H126, E127, V128, K129, V13336GLTKPSLYTMEK11. S39, K55, S57, P59, E60, G61, L62, P63, K65, Y66, K68, E87, G88, G89, P90, G92, S136, M139VKLNPSMNYLSG12. S39, K55, S57, P59, G61, L62, P63, K65, Y66, V67, K68, V85, G88, G89, P90, G92, S136, M139SVTESPNRMGLV13. S39, S57, P59, E60, G61, L62, P63, V67, V85, E87, G88, G89, P90, G92, S136, M139GMSLAQVTAKQN14. S39, K55, S57, G61, L62, K65, V67, K68, V85, G88, G89, G92, Q132, A135, S136, M139SLTNPWPVSFSP15. T7, T9, F30, F58, L62, P63, F64, V67, V85, P90, N118, V133, S136, T142, L143YIPPLPQMLTAI16. I56, P59, L62, P63, Y66, P90, I91, Q132, A135, M139KTASDWMTAQYP17. S39, K55, S57, P59, P63, K65, Y66, K68, P90, Q132, A135, S136, M139LMTKYHIPEISD18. S39, K55, I56, S57, P59, E60, L62, P63, K65, Y66, K68, E87, P90, I91, S136, M13926SNPLNMLRITSL19. S39, S40, I44, I53, I56, S57, L62, P63, R70, I86, P90, I91, L95, S136, M13961.5YSFTPHVDFISP20. D27, F30, P31, S39, S40, V41, I44, I53, I56, S57, F58, P59, F64, Y66, V67, D69, Y83, V85, I86AHNLLYNLKYHP21. N4, Y5, N82, Y83, P90, L95, K97, K115, N118, K119, H121, K134, A135, K137TTDLVLNMLPLS22. D25, D27, N28, P31, V33, P35, S39, S40, V41, S57, P59, D69, M139, T142, L143HPSAENPLTMPP23. E6, T7, P63, P90, S117, N118, A130, E131, A135, S136, E138, M139, E141, T142, L143HPSAENPLTMPP24. S39, S57, P59, E60, L62, P63, E87, P90, A135, S136, M13936VSTPANTKYNNR25. V2, N4, Y5, T7, N82, Y83, V85, P90, K97, S99, K115, S117, N118, K119, V128, V133, K134, A135, S136, K137TSSAPDNWTYWL26. D25, D27, N28, P31, P35, S39, S40, S57, P59, D69, T142, L143, A146, Y150IAAPNSLRFSPL27. S39, I56, S57, F58, P59, L62, P63, F64, P90, I91, A135, S136TLTSAPLKWTPR28. S39, S40, K54, K55, S57, L62, P63, K65, K68, R70, P90, L95, K97, A135, S13636HLISSNKPQHTQ61. N4, T7, N82, P90, L95, K97, S99, I102, K115, I116, S117, N118, K119, H121, Q132, K134, S136, K13762.5WNISSAKPMLPY62. S39, S40, I44, I53, K54, K55, I56, S57, L62, P63, K65, Y66, K68, Y83, I86, P90, I91, L95, K97, A135, S136, M139WNISSAKPMLPY63. S39, K55, I56, S57, P59, L62, P63, K65, Y66, K68, P90, I91, A135, S136, M139GMLSSLKPPIAH64. S39, K55, I56, S57, P59, G61, L62, P63, K65, K68, G88, G89, P90, I91, G92, A135, S136, M139SINAWVPWLIDS65. I23, D25, D27, N28, P31, V33, P35, S39, S40, V41, I56, S57, P59, D69, L143, A146SWMTSFKHSANS66. F3, N4, T7, N82, K97, S99, K115, S117, N118, K119, H121, K134, A135, S136, K137, M139AFMSSAKLPDSR67. S39, K55, S57, F58, P59, L62, P63, F64, K65, K68, P90, A135, S136, M139SSAAMLHSTKSL68. T7, L95, K97, S99, K115, S117, K119, H121, K134, A135, S136, K137, M13936ESIHASGKMGRH69. S39, K55, I56, S57, E60, G61, K65, K68, E87, G88, G89, I91, G92, A135, S136, M139ANHLSGNNYGIS70. S39, S40, I44, I53, I56, S57, L62, Y66, Y83, I86, G88, G89, I91, G92, L95, A135, S136ANHLSGNNYGIS71. N43, I44, G48, G49, G51, I53, I56, S57, Y66, Y81, N82, Y83, I86, G88, G89, I91, S99GTTTLNHNYSAK72. N4, Y5, T7, N82, Y83, L95, K97, S99, K115, S117, N118, K119, H121, K134, A135, S136, K137, G140GTTTLNHNYSAK73. N4, Y5, T7, T9, K80, Y81, N82, Y83, S99, K103, K115, S117, N118, K119, S136, K137, G140, L143YHPNGMNPYTKA74. K55, P59, G61, P63, K65, Y66, K68, G88, G89, P90, G92, A135, M139NDNYPATIKHSN75. I23, D27, P50, I53, K54, I56, K68, D69, D72, K80, Y81, N82, Y83, I86, K97, S99, I102, I116, S117, N118, K119SVDMPHPYGQRP76. S39, S40, V41, S57, P63, Y66, V67, D69, R70, Y83, V85, G88, G89, P90, G92, S136, M13946WNQGSPNLRYTS40. T7, T9, Q36, L62, P63, G89, P90, N118, Q132, S136, G140, T142, L143, R14553.8WNQGSPNLRYTS41. S39, S40, S57, L62, P63, Y66, R70, Y83, G88, G89, P90, G92, L95, S136VSTAKYETLNEQ42. N4, Y5, E6, T7, E8, T9, K80, Y81, N82, Y83, S99, E101, K103, V105, K115, S117, N118, K119, V133, S136, K137, E141, L143VSTAKYETLNEQ43. V2, N4, Y5, E6, T7, N82, Y83, V85, L95, K97, S99, E101, K115, S117, N118, K119, V128, Q132, V133, K134, A135, S136, K137VPTECSGRTSCT44. G1, V2, V67, V85, E87, G88, G89, P90, G92, T94, E96, S99, T122, G124, E127, V128, V133DPLDQGKMLRTP45. K55, P59, G61, L62, P63, K65, K68, G88, G89, P90, G92, Q132, M139SIPRYYHTENSP46. S39, S40, E42, I44, I53, I56, S57, P63, Y66, R70, Y83, I86, E87, P90, I91, S13636SIPRYYHTENSP47. I23, P50, I53, I56, R70, E73, Y81, N82, Y83, I86, S99, E101, I102, I116, S117, N118SIPRYYHTENSP48. N28, P31, S40, E42, N43, I44, E45, N47, I53, I56, S57, Y66, R70, Y83, I86, E87SSPSAAQWLNIG49. S39, I56, S57, P59, G61, L62, P63, G88, G89, P90, I91, G92, Q132, A135, S136LPGRAHDPWKVP50. V41, K54, K55, L62, P63, K65, V67, K68, D69, R70, V85, G88, G89, P90, G92, L95, K97, A135HPATSNTKYWIN51. S39, S40, I44, I53, K54, K55, I56, S57, P63, K65, Y66, K68, Y83, I86, P90, I91, K97, A135, S136HPATSNTKYWIN52. N4, Y5, T7, N82, Y83, P90, K97, S99, I102, K115, I116, S117, N118, K119, H121, K134, A135, S136, K137FTTESGGLNPHA53. S39, S57, F58, P59, E60, G61, L62, P63, F64, E87, G88, G89, P90, G92, A135, S136VEAHNQWTGLVA54. G1, V2, N4, G92, T94, L95, E96, N118, H121, T122, H126, E127, V128, A130, E131, Q132, V133, A135YHPNGMNPYTKA55. K55, P59, G61, P63, K65, Y66, K68, G88, G89, P90, G92, A135, M139APTIAYNTFMPM56. I44, I53, I56, F58, P63, F64, Y66, Y83, I86, P90, I91, A135, M139APTIAYNTFMPM57. T9, F22, I23, F30, P31, P35, I56, I102, I116, M139, T142, A146, Y150APTIAYNTFMPM58. F22, I23, N28, F30, P31, P35, I56, F58, P59, M139, T142, A146, Y150APTIAYNTFMPM59. I56, F58, P59, P63, F64, Y66, P90, I91, A135, M139APTIAYNTFMPM60. F30, P31, N43, I44, I53, I56, F58, P59, F64, Y66, Y83, I86, M139IYGQVLSTVQYR29. S39, I56, S57, G61, L62, Y66, V67, V85, G88, G89, I91, G92, Q132, S136DLTFTVNPLSKA30. S39, K55, S57, F58, P59, L62, P63, F64, K65, V67, K68, V85, P90, A135, S13656STIAERSIHPFN31. S39, I56, S57, F58, P59, E60, P63, F64, E87, P90, I91, A135, S13636.8NLSTGEKPEVTK32. S39, K55, S57, P59, E60, G61, L62, P63, K65, V67, K68, V85, E87, G88, G89, P90, G92, S136SLSSMNTIRVPG33. S39, I56, S57, P59, G61, L62, P63, V67, V85, G88, G89, P90, I91, G92, S136, M139HGVMTVSTTEKF34. S39, K55, S57, F58, E60, G61, F64, K65, V67, K68, V85, E87, G88, G89, G92, S136, M139HMGMTKINYSAL35. S39, K55, I56, S57, G61, L62, K65, Y66, K68, G88, G89, I91, G92, A135, S136, M139LSSHKATSTRAD36. D93, T94, L95, K119, H121, T122, K123, H126, K129, A130, K134, A135, S13636LSSHKATSTRAD37. R17, D25, K32, L143, R145, A146, S149, L152, A153, H154, S155, D156, A157SLPFGPNTTIRP38. S39, I56, S57, F58, P59, G61, L62, P63, F64, G88, G89, P90, I91, G92, S136LESHYTQASYTQ39. T9, T10, E141, T142, L143, A146, E148, S149, Y150, L152, A153, H154, A157, Y158Identical peptides in the same individual (boldface) or in different patients (italics) are indicated. Open table in a new tab Identical peptides in the same individual (boldface) or in different patients (italics) are indicated.

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