Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens

Abstract

Background: Patients with peanut allergy have highly stable pathologic antibody repertoires to the immunodominant B-cell epitopes of the major peanut allergens Ara h 1 to 3.

Objective: We used a peptide microarray technique to analyze the effect of treatment with peanut oral immunotherapy (OIT) on such repertoires.

Methods: Measurements of total peanut-specific IgE (psIgE) and peanut-specific IgG(4) (psIgG(4)) were made with CAP-FEIA. We analyzed sera from 22 patients with OIT and 6 control subjects and measured serum specific IgE and IgG(4) binding to epitopes of Ara h 1 to 3 using a high-throughput peptide microarray technique. Antibody affinity was measured by using a competitive peptide microarray, as previously described.

Results: At baseline, psIgE and psIgG(4) diversity was similar between patients and control subjects, and there was broad variation in epitope recognition. After a median of 41 months of OIT, polyclonal psIgG(4) levels increased from a median of 0.3 μg/mL (interquartile range [25% to 75%], 0.1-0.43 μg/mL) at baseline to 10.5 μg/mL (interquartile range [25% to 75%], 3.95-45.48 μg/mL; P < .0001) and included de novo specificities. psIgE levels were reduced from a median baseline of 85.45 kU(A)/L (23.05-101.0 kU(A)/L) to 7.75 kU(A)/L (2.58-30.55 kU(A)/L, P < .0001). Affinity was unaffected. Although the psIgE repertoire contracted in most OIT-treated patients, several subjects generated new IgE specificities, even as the total psIgE level decreased. Global epitope-specific shifts from IgE to IgG(4) binding occurred, including at an informative epitope of Ara h 2.

Conclusion: OIT differentially alters Ara h 1 to 3 binding patterns. These changes are variable between patients, are not observed in control subjects, and include a progressive polyclonal increase in IgG(4) levels, with concurrent reduction in IgE amount and diversity.

Figure 1

OIT alters the peanut-specific immunoglobulin response in quantity and intensity. In these figures, each data point represents one subject. Lines or plus signs indicate medians. (A) Peanut-specific IgE, as measured by conventional CAP-FEIA, decreases from a median of 85.5 kU_A/L at baseline to 7.8 kU_A/L after OIT treatment (p<0.0001). (B) The overall IgE repertoire remains stable even as psIgE decreases. All array spots with a Z-score > 3 were considered positive. (C) IgE level and epitope diversity are strongly correlated at baseline and at the time of the last measurement, after a median of 41 months of treatment. (D) Fewer peanut peptides are bound by IgE with high intensity over the course of OIT treatment.

Figure 1

OIT alters the peanut-specific immunoglobulin response in quantity and intensity. In these figures, each data point represents one subject. Lines or plus signs indicate medians. (A) Peanut-specific IgE, as measured by conventional CAP-FEIA, decreases from a median of 85.5 kU_A/L at baseline to 7.8 kU_A/L after OIT treatment (p<0.0001). (B) The overall IgE repertoire remains stable even as psIgE decreases. All array spots with a Z-score > 3 were considered positive. (C) IgE level and epitope diversity are strongly correlated at baseline and at the time of the last measurement, after a median of 41 months of treatment. (D) Fewer peanut peptides are bound by IgE with high intensity over the course of OIT treatment.

Figure 1

OIT alters the peanut-specific immunoglobulin response in quantity and intensity. In these figures, each data point represents one subject. Lines or plus signs indicate medians. (A) Peanut-specific IgE, as measured by conventional CAP-FEIA, decreases from a median of 85.5 kU_A/L at baseline to 7.8 kU_A/L after OIT treatment (p<0.0001). (B) The overall IgE repertoire remains stable even as psIgE decreases. All array spots with a Z-score > 3 were considered positive. (C) IgE level and epitope diversity are strongly correlated at baseline and at the time of the last measurement, after a median of 41 months of treatment. (D) Fewer peanut peptides are bound by IgE with high intensity over the course of OIT treatment.

Figure 1

OIT alters the peanut-specific immunoglobulin response in quantity and intensity. In these figures, each data point represents one subject. Lines or plus signs indicate medians. (A) Peanut-specific IgE, as measured by conventional CAP-FEIA, decreases from a median of 85.5 kU_A/L at baseline to 7.8 kU_A/L after OIT treatment (p<0.0001). (B) The overall IgE repertoire remains stable even as psIgE decreases. All array spots with a Z-score > 3 were considered positive. (C) IgE level and epitope diversity are strongly correlated at baseline and at the time of the last measurement, after a median of 41 months of treatment. (D) Fewer peanut peptides are bound by IgE with high intensity over the course of OIT treatment.

Figure 2

OIT increases the production of broadly diverse IgG4 antibodies, some of which have de novo specificity. (A) A polyclonal expansion of IgG4 production occurs while on OIT but not an allergen elimination diet. Plus signs indicate median values. (B) The specificity of the IgG4 response after treatment includes both peptides recognized before OIT and new specificities. Each circle or diamond represents one subject.

Figure 2

OIT increases the production of broadly diverse IgG4 antibodies, some of which have de novo specificity. (A) A polyclonal expansion of IgG4 production occurs while on OIT but not an allergen elimination diet. Plus signs indicate median values. (B) The specificity of the IgG4 response after treatment includes both peptides recognized before OIT and new specificities. Each circle or diamond represents one subject.

Figure 3

Scatter plot comparing IgG4 (A) and IgE (B) binding to the array between the standard protocol (x-axis) and the competition assay (y-axis) from baseline and last time points of 5 subjects. Each spot represents antibody binding (represented in Z-score) to one peanut peptide on the array.

Figure 4

The peanut IgE repertoire contracts in most but not all subjects treated with OIT. The number of array spots bound by IgE was compared from baseline to the last available measurement for each subject.

Figure 5

Comparison of IgE and IgG4 binding of peanut OIT treated patients to peptides of Ara h 1-3 between baseline and last time point. The x-axis shows the overlapping peptides. The left y-axis shows the average IgE and IgG4 binding level (represented as Z-scores) to each peptide. The right y-axis shows the difference in the percentage of patients showing positive binding to each peptide between time points. Binding for IgE is indicated above the x-axis line while binding for IgG4 is below the line. Regions/peptides with significantly (p<0.01, FDR<0.1, identified using TileMap) higher binding at baseline than last time point are indicated with red triangles, while regions with significantly higher binding at last time point than baseline are indicated with blue triangles. Green boxes labeled A-F indicate regions where varied concordance of IgE and IgG4 binding is of interest.