Epitope-Specific Neutralizing IgG4 Antibodies Define Natural and Acquired Tolerance in Peanut Allergy

Introduction/Rationale: While early introduction has clinically transformed our care of food allergy patients by inducing long-lived natural tolerance in children, the underlying mechanism of natural tolerance remains unclear, though serum IgG4 levels have been shown to increase in tolerant and peanut consuming adolescents. In oral immunotherapy (OIT), neutralizing IgG4 antibodies have been associated with sustained tolerance. We hypothesize that neutralizing IgG4 antibodies (nAbs) are also induced in peanut-allergic individuals with high thresholds of reactivity.

Methods: To study this, we developed three biomarker assays: an indirect ELISA which was previous benchmarked in tolerance after OIT, an indirect bead-based assay (iBBNA), and a direct bead-based assay (dBBNA). The ELISA and iBBNA using Ara h 2 specific epitope-specific nAbs to epitopes 1.2 and 3 that block neutralizing epitopes on Ara h 2. The dBBNA uses a mutant Ara h 2 that preserves epitopes 1.2 and 3 and therefore measures nAb levels. Protein G purified serum from peanut-allergic individuals with a low ( < 144 mg, n=10) or high reactivity (>144mg, n= 10) peanut were used for profiling.

Results: Naturally tolerant individuals exhibited robust neutralizing IgG4 responses, mirrored by OIT subjects achieving sustained unresponsiveness, but absent in transiently desensitized patients. The iBBNA, validated against inhibitory ELISA (R² = 0.9, CV = 2.5%), reliably distinguished durable from transient OIT responses.

Conclusion: These findings establish neutralizing IgG4 as a shared feature of natural and therapeutic tolerance, supporting their use as predictive biomarkers and guiding hypo-allergen design.