Epicutaneous immunotherapy for peanut allergy modifies IgG4 responses to major peanut allergens
Authors: Stef J. Koppelman, Aurélie Peillon, Wenceslas Agbotounou, Hugh A. Sampson, Laurent Martin
Journal: Journal of Allergy and Clinical Immunology (2018)

To date, no therapy for peanut allergy is available. Several therapeutic options are under investigation and show promise. Epicutaneous immunotherapy (EPIT) for peanut allergy has been shown to increase significantly the threshold levels of peanut-allergic patients, in particular in children.1Sampson H.A. Shreffler W.G. Yang W.H. Sussman G.L. Brown-Whitehorn T.F. Nadeau K.C. et al.Effect for varying doses of epicutaneous immunotherapy vs placebo on reaction to peanut protein exposure among patients with peanut sensitivity.JAMA. 2017; 318: 1798-1809Crossref PubMed Scopus (78) Google Scholar The proposed mechanism of EPIT is that allergens are rapidly taken up by skin antigen-presenting cells and transported to regional lymph nodes, thereby inducing a humoral and cellular immunological response.2Mondoulet L. Dioszeghy V. Ligouis M. Dhelft V. Dupont C. Benhamou P.H. Epicutaneous immunotherapy on intact skin using a new delivery system in a murine model of allergy.Clin Exp Allergy. 2010; 40: 659-667Crossref PubMed Scopus (125) Google Scholar In a recent phase 2b clinical trial, several doses of peanut EPIT were evaluated in children, adolescents, and adults aiming to increase the patient's threshold of reactivity to peanut. The primary end point of the study was met, and the largest treatment effect was observed in children (6-11 years).1Sampson H.A. Shreffler W.G. Yang W.H. Sussman G.L. Brown-Whitehorn T.F. Nadeau K.C. et al.Effect for varying doses of epicutaneous immunotherapy vs placebo on reaction to peanut protein exposure among patients with peanut sensitivity.JAMA. 2017; 318: 1798-1809Crossref PubMed Scopus (78) Google Scholar Using samples from the study reported by Sampson et al,1Sampson H.A. Shreffler W.G. Yang W.H. Sussman G.L. Brown-Whitehorn T.F. Nadeau K.C. et al.Effect for varying doses of epicutaneous immunotherapy vs placebo on reaction to peanut protein exposure among patients with peanut sensitivity.JAMA. 2017; 318: 1798-1809Crossref PubMed Scopus (78) Google Scholar the current report evaluates the serological changes in children treated with either a peanut patch (250 μg peanut protein, referred to as 250-μg patch; n = 25) or a placebo patch (n = 26). Similar to the protein composition of peanut,3Koppelman S.J. Jayasena S. Luykx D. Schepens E. Apostolovic D. de Jong G.A. et al.Allergenicity attributes of different peanut market types.Food Chem Toxicol. 2016; 91: 82-90Crossref PubMed Scopus (21) Google Scholar the 250-μg patch contains small amounts of Ara h 6 and Ara h 2, larger amounts of Ara h 1, while Ara h 3 is the most abundant protein (see Table E1 in this article's Online Repository at www.jacionline.org). The report by Sampson et al1Sampson H.A. Shreffler W.G. Yang W.H. Sussman G.L. Brown-Whitehorn T.F. Nadeau K.C. et al.Effect for varying doses of epicutaneous immunotherapy vs placebo on reaction to peanut protein exposure among patients with peanut sensitivity.JAMA. 2017; 318: 1798-1809Crossref PubMed Scopus (78) Google Scholar showed that treatment with the 250-μg patch induced IgG4 to total peanut protein, and in this study, we sought to investigate the specificity of the IgG4 induction by assessing IgG4 to peanut components following treatment. Sera from subjects in the children treatment groups were collected at both baseline and several time points during EPIT treatment. Two testing platforms were used to analyze immunoglobulins. First, ImmunoCAP (Thermo Fisher Scientific, Uppsala, Sweden) was used to obtain quantitative results for peanut components Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9 (results expressed in kUA/L for IgE and in mgA/L for IgG4). Ara h 6 was not available as reagent for ImmunoCAP. Second, Immuno Solid-phase Allergy Chip (ISAC; Thermo Fisher Scientific) was used because this also contains Ara h 6 in addition to the components available for ImmunoCAP. A disadvantage of ISAC is that the results are semi-quantitative (results expressed in ISAC Standardized Units as defined by the manufacturer) and cannot be directly compared with ImmunoCAP. Baseline sensitization of subjects to peanut components was evaluated by ImmunoCAP and ISAC. IgE determined by ISAC and ImmunoCAP correlated well for allergens available on both platforms (see Fig E1 in this article's Online Repository at www.jacionline.org). Table I presents the baseline sensitization of the 51 children in the 2 groups (250-μg patch and placebo patch). No differences were observed between the 2 treatment groups (P > .6, Wilcoxon rank-sum test). As determined by ISAC, all subjects were sensitized to Ara h 2 and Ara h 6, with a high median IgE titer. Ara h 1 and Ara h 3 were frequently recognized but with lower IgE titers than observed for Ara h 2 or Ara h 6 (Table I). Ara h 8 and Ara h 9 were least frequently recognized with low IgE titers. IgE titers to Ara h 2 and Ara h 6 were highly correlated (Fig 1; R2 = 0.797), but several patients had high IgE titers to Ara h 2 while not to Ara h 6, or vice versa. Overall, the IgE titers for Ara h 6 were slightly lower than for Ara h 2. Close correlation between IgE to Ara h 2 and IgE to Ara h 6 is expected on the basis of their homology and partially shared IgE epitopes4Koppelman S.J. de Jong G.A. Laaper-Ertmann M. Peeters K.A. Knulst A.C. Hefle S.L. et al.Purification and immunoglobulin E-binding properties of peanut allergen Ara h 6: evidence for cross-reactivity with Ara h 2.Clin Exp Allergy. 2005; 35: 490-497Crossref PubMed Scopus (116) Google Scholar and is in line with an earlier report that used an experimental Ara h 6 ImmunoCAP showing correlation between concentrations of IgE to Ara h 2 and Ara h 6.5Ballmer-Weber B.K. Lidholm J. Fernández-Rivas M. Seneviratne S. Hanschmann K.M. Vogel L. et al.IgE recognition patterns in peanut allergy are age dependent: perspectives of the EuroPrevall study.Allergy. 2015; 70: 391-407Crossref PubMed Scopus (92) Google ScholarTable IBaseline sensitization profile of study subjects randomized in the 250-μg patch group, the placebo group, and for combined groupsGroupPeanutAra h 1Ara h 2Ara h 3Ara h 6Ara h 8Ara h 9CAPCAPISACCAPISACCAPISACCAPISACCAPISACCAPISACViaskin 250 μg n positive25 of 2521 of 2523 of 2524 of 2525 of 2515 of 2517 of 25NA25 of 256 of 259 of 251 of 250 of 25 Median55.024.38.333.836.73.33.0NA25.60.40.00.40.0 LQ20.50.81.511.512.40.40.0NA8.70.40.00.40.0 UQ217.370.818.276.751.822.724.6NA30.60.40.70.40.0Placebo n positive26 of 2618 of 2621 of 2624 of 2626 of 2614 of 2619 of 26NA26 of 269 of 259 of 252 of 260 of 26 Median89.87.710.338.821.70.82.3NA25.10.40.20.40.0 LQ17.80.41.75.712.00.40.0NA7.20.40.00.40.0 UQ284.657.430.094.256.721.424.8NA40.00.60.50.40.0All n positive51 of 5139 of 5144 of 5148 of 5151 of 5129 of 5136 of 51NA51 of 5115 of 5115 of 513 of 510 of 51 Median55.011.39.734.229.51.33.0NA25.50.40.00.40.0 LQs17.80.61.56.812.00.40.0NA8.50.40.00.40.0 UQ284.667.323.494.256.722.724.8NA36.50.60.70.40.0CAP, ImmunoCAP; LQ, lower quartile; NA, not available (allergen Ara h 6 is not available as ImmunoCAP reagent); Peanut, total peanut extract; UQ, upper quartile. Open table in a new tab CAP, ImmunoCAP; LQ, lower quartile; NA, not available (allergen Ara h 6 is not available as ImmunoCAP reagent); Peanut, total peanut extract; UQ, upper quartile. Baseline sera and sera obtained during treatment were tested for peanut allergen-specific IgG4 using ImmunoCAP and ISAC. Fig E1, D, shows that the 2 assays correlate well for those peanut components available on both assays. Fig 2 shows the induction of IgG4 per subject per peanut component for subjects treated with the 250-μg patch. Subjects showed an increase in IgG4 for Ara h 1, Ara h 2, Ara h 3, and Ara h 6, although the extent of this increase varied among subjects. For all subjects, Ara h 2 and Ara h 6 induced the highest levels of IgG4. After 12 months of treatment, using ImmunoCAP, the increase in median IgG4 for Ara h 2 was 62.7-fold, whereas Ara h 1 and Ara h 3 showed a smaller fold increase in median of 9.5- and 2.4-fold, respectively. The median IgG4 levels for Ara h 8 and Ara h 9 did not change over time. ISAC data show that Ara h 6 induces IgG4 to a comparable level as Ara h 2, and stronger than do the other peanut allergens (Fig 2). Ara h 2 and Ara h 6 are the strongest inducers of IgG4 even though these allergens are present on the patch in lower amounts than Ara h 1 and Ara h 3 (see Table E1). Of note, in patients with a high IgE level for Ara h 2 and/or Ara h 6 at baseline, the induction of IgG4 to these allergens was greater than in patients with low IgE level for Ara h 2 and/or Ara h 6 at baseline. However, the number of patients, especially those with low IgE level to Ara h 2 and/or Ara h 6, is too low to prove correlation (Pearson correlation coefficients [r] 0.61, 0.56, and 0.47, for Ara h 2 [baseline IgE by ImmunoCAP], Ara h 2 [baseline IgE by ISAC], and Ara h 6 [baseline IgE by ISAC] and corresponding P values of .0011, .0038, and .0174, respectively; data not shown). In the placebo-treated patients, no increase in IgG4 was observed. In this group, the baseline IgG4 levels were comparable to those of the 250-μg patch–treated group (Fig 2, A), with medians of 0.07, 0.17, 0.15, 0.07, and 0.07 mgA/L, respectively, for Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9. These levels did not change during the course of the study (medians at 12 months of placebo treatment for the respective peanut allergens were 0.07, 0.17, 0.14, 0.07, and 0.07 mgA/L), in line with the noted absence of IgG4 induction for total peanut in this group.1Sampson H.A. Shreffler W.G. Yang W.H. Sussman G.L. Brown-Whitehorn T.F. Nadeau K.C. et al.Effect for varying doses of epicutaneous immunotherapy vs placebo on reaction to peanut protein exposure among patients with peanut sensitivity.JAMA. 2017; 318: 1798-1809Crossref PubMed Scopus (78) Google Scholar The combined results show that peanut EPIT strongly induces IgG4 to Ara h 2 and Ara h 6, which are known as the most important peanut allergens,6Zhuang Y. Dreskin S.C. Redefining the major peanut allergens.Immunol Res. 2013; 55: 125-134Crossref PubMed Scopus (59) Google Scholar and to a lesser extent to Ara h 1 and Ara h 3. Baseline sera and sera obtained during treatment were also tested for peanut allergen-specific IgE, using ImmunoCAP. Fig E2 in this article's Online Repository at www.jacionline.org shows an increase of about 2-fold during the first half year of EPIT, followed by a decrease at 1 year of EPIT treatment. This was observed for Ara h 1, Ara h 2, and Ara h 3 (Ara h 6 not available on ImmunoCAP), while for Ara h 8 and Ara h 9 the median IgE levels were low and no clear trend could be observed. Initial increase in IgE followed by a decrease at 1 year of treatment is often seen in allergen immunotherapy, and this was also described for total peanut IgE in this current treatment population.1Sampson H.A. Shreffler W.G. Yang W.H. Sussman G.L. Brown-Whitehorn T.F. Nadeau K.C. et al.Effect for varying doses of epicutaneous immunotherapy vs placebo on reaction to peanut protein exposure among patients with peanut sensitivity.JAMA. 2017; 318: 1798-1809Crossref PubMed Scopus (78) Google Scholar Induction of IgG4 has been described for other investigational therapies for peanut allergy. Oral immunotherapy (OIT) treatment showed induction of IgG4 to Ara h 1 and Ara h 2, and to a lesser extent to Ara h 3.7Vickery 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 (154) Google Scholar, 8Nozawa A. Okamoto Y. Movérare R. Borres M.P. Kurihara K. Monitoring Ara h 1, 2 and 3-sIgE and sIgG4 antibodies in peanut allergic children receiving oral rush immunotherapy.Pediatr Allergy Immunol. 2014; 25: 323-328Crossref PubMed Scopus (34) Google Scholar The strongest response was found for Ara h 2, with an increase in median compared with baseline of about 25-fold after 1 year of treatment.8Nozawa A. Okamoto Y. Movérare R. Borres M.P. Kurihara K. Monitoring Ara h 1, 2 and 3-sIgE and sIgG4 antibodies in peanut allergic children receiving oral rush immunotherapy.Pediatr Allergy Immunol. 2014; 25: 323-328Crossref PubMed Scopus (34) Google Scholar A similar pattern of IgG4 induction was found with sublingual immunotherapy (SLIT), with Ara h 2 again being the strongest IgG4 inducer, showing an increase in median of about 10-fold compared with baseline.9Burk C.M. Kulis M. Leung N. Kim E.H. Burks A.W. Vickery B.P. Utility of component analyses in subjects undergoing sublingual immunotherapy for peanut allergy.Clin Exp Allergy. 2016; 46: 347-353Crossref PubMed Scopus (19) Google Scholar The OIT and SLIT studies did not evaluate IgG4 responses to Ara h 6. The fold increases in component-specific IgG4 achieved by EPIT shown here are comparable to those obtained with OIT8Nozawa A. Okamoto Y. Movérare R. Borres M.P. Kurihara K. Monitoring Ara h 1, 2 and 3-sIgE and sIgG4 antibodies in peanut allergic children receiving oral rush immunotherapy.Pediatr Allergy Immunol. 2014; 25: 323-328Crossref PubMed Scopus (34) Google Scholar and SLIT.9Burk C.M. Kulis M. Leung N. Kim E.H. Burks A.W. Vickery B.P. Utility of component analyses in subjects undergoing sublingual immunotherapy for peanut allergy.Clin Exp Allergy. 2016; 46: 347-353Crossref PubMed Scopus (19) Google Scholar In terms of absolute amounts of IgG4 as determined with ImmunoCAP, we show that 12 months of peanut EPIT results in median concentrations of 0.76, 7.52, and 0.33 mgA/L for Ara h 1, Ara h 2, and Ara h 3, respectively. These concentrations are similar, or slightly higher in the case of Ara h 2, to those found after 12 months of OIT8Nozawa A. Okamoto Y. Movérare R. Borres M.P. Kurihara K. Monitoring Ara h 1, 2 and 3-sIgE and sIgG4 antibodies in peanut allergic children receiving oral rush immunotherapy.Pediatr Allergy Immunol. 2014; 25: 323-328Crossref PubMed Scopus (34) Google Scholar or SLIT.9Burk C.M. Kulis M. Leung N. Kim E.H. Burks A.W. Vickery B.P. Utility of component analyses in subjects undergoing sublingual immunotherapy for peanut allergy.Clin Exp Allergy. 2016; 46: 347-353Crossref PubMed Scopus (19) Google Scholar These changes in IgG4 were observed with treatment using only 250 μg of peanut protein (∼1/1000 of 1 peanut) applied epicutaneously daily. In conclusion, our study demonstrates that EPIT for peanut allergy induces IgG4 to major peanut allergens, comparable in quantity and specificity to other investigational treatment modalities for peanut allergy. Furthermore, our study shows that Ara h 6 is an important allergen in terms of sensitization and that EPIT induces a strong IgG4 response to this allergen. Table E1 presents the allergen levels in extracts used to manufacture the 250-μg patch. Ara h 3 is the most abundant protein, followed by Ara h 1. Ara h 2 and Ara h 6 represent a minor part of the total protein amount, together about 8% to 15% of all protein. These allergen levels are highly comparable with those found in extracts described in literature (Table E1, right column).E1Koppelman S.J. Jayasena S. Luykx D. Schepens E. Apostolovic D. de Jong G.A. et al.Allergenicity attributes of different peanut market types.Food Chem Toxicol. 2016; 91: 82-90Crossref PubMed Scopus (38) Google ScholarTable E1Global protein composition of extracts used for the production of the 250-μg patchAllergenRange (% of total protein)Extracts for patch (n = 13)Extracts from literature (n = 20)E1Koppelman S.J. Jayasena S. Luykx D. Schepens E. Apostolovic D. de Jong G.A. et al.Allergenicity attributes of different peanut market types.Food Chem Toxicol. 2016; 91: 82-90Crossref PubMed Scopus (38) Google ScholarAra h 119-2812-24Ara h 25-94-8Ara h 362-7258-84Ara h 63-63-10 Open table in a new tab Baseline sera (n = 51) were tested for IgE titers for peanut components using both ImmunoCAP and ISAC. ImmunoCAP is available for Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9, whereas ISAC also provides Ara h 6. Fig E1, A, shows the correlation between the sum of IgE titers determined by ImmunoCAP and ISAC, the latter with Ara h 6 IgE included (R2 = 0.900) or excluded (R2 = 0.904). Correlation between IgE to total peanut in ImmunoCAP (ImmunoCAP F13) and the sum of IgE levels to peanut components measured by ImmunoCAP (R2 = 0.964) and ISAC (R2 = 0.895) is shown in Fig E1, B. For individual components present on both platforms, the correlation is shown in Fig E1, C (Ara h 1: R2 = 0.784; Ara h 2: R2 = 0.876; Ara h 3: R2 = 0.922; Ara h 8: R2 = 0.792; Ara h 9: R2 < 0.005). For IgE, ImmunoCAP and ISAC correlated well, except for Ara h9 for which we found in some sera IgE by ImmunoCAP, but no IgE by ISAC. For components considered as most important allergens (Ara h 2 and Ara h 1), IgE determination of both methods correlate well. Sera obtained after 12 months of EPIT with 250-μg patch (n = 25) were analyzed for IgG4 to peanut components using both ImmunoCAP and ISAC. ImmunoCAP is available for Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9, whereas ISAC also provides Ara h 6. Fig E1, D, shows the correlation between the 2 methods for components present for both methods (Ara h 1: R2 = 0.863; Ara h 2: R2 = 0.737; Ara h 3: R2 = 0.7433; Ara h 8: R2 = 0.896; Ara h 9: R2 = 0.908). For IgG4, ImmunoCAP and ISAC correlated well for all peanut components. Sera from subjects on active EPIT treatment (250-μg patch) from time points 0, 3, 6, and 12 months were analyzed for IgE to individual peanut components Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9 using ImmunoCAP (Fig E2). IgE to Ara h 6 was not tested because Ara h 6 is not available on ImmunoCAP. ISAC could not be used to follow IgE evolution during EPIT, because of the disturbing effect of IgG on ISAC. For the main peanut allergens Ara h 1, Ara h 2, and Ara h 3, the IgE titer increased initially and decreased again at 12 months of EPIT, in line with what is often observed for allergen immunotherapy and what was described for the patient population for total peanut IgE.E2Sampson H.A. Shreffler W.G. Yang W.H. Sussman G.L. Brown-Whitehorn T.F. Nadeau K.C. et al.Effect for varying doses of epicutaneous immunotherapy vs placebo on reaction to peanut protein exposure among patients with peanut sensitivity.JAMA. 2017; 318: 1798-1809Crossref PubMed Scopus (137) Google Scholar