IgE and IgG4 epitopes of the peanut allergens shift following oral immunotherapy

Background Oral immunotherapy (OIT) with peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Aimmune Therapeutics) is an FDA-approved treatment to desensitize peanut allergic participants. Objective Here we assessed shifts in IgE and IgG4 binding to peanut allergens and their epitopes recognized by United States (US) peanut allergic participants (n = 20) enrolled in phase 3 PTAH OIT clinical trials. Methods Pre- and post- trial participant sera were collected approximately 12 months apart and tested for IgE binding to intact peanut proteins via ImmunoCAP ISAC immunoassays. IgE and IgG4 linear epitopes were identified based on binding to synthetic overlapping 15-mer linear peptides of 10 peanut allergens (Ara h 1-11) synthesized on microarray slides. Results Statistically significant decreases in IgE binding were identified for intact Ara h 2, 3, and 6, and known and newly identified IgE epitopes were shown to exhibit shifts towards IgG4 binding post-OIT, with most linear peptides having increased IgG4 binding after treatment with PTAH. While PTAH does not seem to alter the actual peptide binding patterns significantly after one year of treatment, the IgE and IgG4 binding ratios and intensity are altered. Conclusion At a population level, the linear IgE and IgG4 epitopes of 10 peanut allergens overlap and that increase in IgG4 with OIT results in displacement of IgE binding to both conformational and linear epitopes. Furthermore, it appears as though the increase in IgG4 is more important to achieve desensitization at the 12-month timepoint than the decrease in IgE. This type of knowledge can be useful in the identification of IgE and IgG4-binding allergen and peptide biomarkers that may indicate desensitization or sustained unresponsiveness of allergic individuals to peanut

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca(2+) o) homeostasis. To elucidate the role of AP2σ2 in Ca(2+) o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue

Purification of Recombinant Peanut Allergen Ara h 1 and Comparison of IgE Binding to the Natural Protein

Allergic reactions to food are on the rise worldwide and there is a corresponding increase in interest to understand the molecular mechanisms responsible. Peanut allergies are the most problematic because the reaction often persists into adulthood and can be as severe as anaphylaxis and death. The purpose of the work presented here was to develop a reproducible method to produce large quantities of pure recombinant Ara h 1(rAra h 1) that will enable standardization of immunological tests for patients and allow structural and immunological studies on the wild type and mutagenized forms of the protein. Ara h 1 is initially a pre-pro-protein which, following two endoproteolytic cleavages, becomes the mature form found in peanut. The mature form however has flexible regions that make it refractory to some structural studies including crystallography. Therefore, independent purification of the mature and core regions was desirable. Expression constructs were synthesized cDNA clones for each in a pET plasmid vector without tags. Codons were optimized for expression in E. coli. High-level expression was achieved in BL21 strains. Purification to near homogeneity was achieved by a combination of ammonium sulfate precipitation and ion exchange chromatography. The purified rAra h 1 was then compared with natural Ara h 1 for IgE binding. All patients recognized both the folded natural and rAra h 1, but the IgE binding to the rArah1 was significantly reduced in comparison to the natural allergen, which could potentially make it useful for immunotherapeutic purposes