Neutralizing IgG4 antibodies are a biomarker of sustained efficacy after peanut oral immunotherapy.

BACKGROUND: Clinical efficacy of oral immunotherapy (OIT) has been associated with the induction of blocking antibodies, particularly those capable of disrupting IgE-allergen interactions. Previously, we identified mAbs to Ara h 2 and structurally characterized their epitopes. OBJECTIVE: We investigated longitudinal changes during OIT in antibody binding to conformational epitopes and correlated the results with isotype and clinical efficacy. METHODS: We developed an indirect inhibitory ELISA using mAbs to block conformational epitopes on immobilized Ara h 2 from binding to serum immunoglobulins from peanut-allergic patients undergoing OIT. We tested the functional blocking ability of mAbs using passive cutaneous anaphylaxis in mice with humanized FcεRI receptors. RESULTS: Diverse serum IgE recognition of Ara h 2 conformational epitopes are similar before and after OIT. Optimal inhibition of serum IgE occurs with the combination of 2 neutralizing mAbs (nAbs) recognizing epitopes 1.2 and 3, compared to 2 nonneutralizing mAbs (non-nAbs). After OIT, IgG4 nAbs, but not IgG1 or IgG2 nAbs, increased in sustained compared to transient outcomes. Induction of IgG4 nAbs occurs after OIT only in those with sustained efficacy. Murine passive cutaneous anaphylaxis after sensitization with pooled human sera is significantly inhibited by nAbs compared to non-nAbs. CONCLUSIONS: Serum IgE conformational epitope diversity remains unchanged during OIT. However, IgG4 nAbs capable of uniquely disrupting IgE-allergen interactions to prevent effector cell activation are selectively induced in OIT-treated individuals with sustained clinical efficacy. Therefore, the induction of neutralizing IgG4 antibodies to Ara h 2 are clinically relevant biomarkers of durable efficacy in OIT.

Short-chain fatty acids (SCFA) in infants' plasma and corresponding mother's milk and plasma in relation to subsequent sensitisation and atopic disease.

BACKGROUND: Short-chain fatty acids (SCFAs) in intestinal contents may influence immune function, while less is known about SCFAs in blood plasma. The aims were to investigate the relation between infants' and maternal plasma SCFAs, as well as SCFAs in mother's milk, and relate SCFA concentrations in infant plasma to subsequent sensitisation and atopic disease. METHODS: Infant plasma (N = 148) and corresponding mother's milk and plasma were collected four months postpartum. Nine SCFA (formic, acetic, propionic, isobutyric, butyric, succinic, valeric, isovaleric, and caproic acid) were analysed by UPLC-MS. At 12 months of age, atopic disease was diagnosed by a pediatric allergologist, and sensitisation was measured by skin prick test. All families participated in the Swedish birth cohort NICE (Nutritional impact on Immunological maturation during Childhood in relation to the Environment). FINDINGS: Infants with sensitisation, atopic eczema, or food allergy had significantly lower concentrations of five, three, and two SCFAs, respectively, in plasma at four months. Logistic regressions models showed significant negative associations between formic, succinic, and caproic acid and sensitisation [ORadj (95% CI) per SD: 0.41 (0.19-0.91); 0.19 (0.05-0.75); 0.25 (0.09-0.66)], and between acetic acid and atopic eczema [0.42 (0.18-0.95)], after adjusting for maternal allergy. Infants' and maternal plasma SCFA concentrations correlated strongly, while milk SCFA concentrations were unrelated to both. Butyric and caproic acid concentrations were enriched around 100-fold, and iso-butyric and valeric acid around 3-5-fold in mother's milk, while other SCFAs were less prevalent in milk than in plasma. INTERPRETATION: Butyric and caproic acid might be actively transported into breast milk to meet the needs of the infant, although mechanistic studies are needed to confirm this. The negative associations between certain SCFAs on sensitisation and atopic disease adds to prior evidence regarding their immunoregulatory potential. FUNDING: Swedish Research Council (Nr. 2013-3145, 2019-0137 and 2023-02217 to A-S.S.), Swedish Research Council for Health, Working Life and Welfare FORTE, Nr 2018-00485 to A.W.), The Swedish Asthma and Allergy Association's Research Fund (2020-0020 to A.S.).

Design of an Ara h 2 hypoallergen from conformational epitopes.

INTRODUCTION: Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapy suggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). METHODS: X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. RESULTS: A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. CONCLUSION: These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.