A phase 1 study of heat/phenol-killed, E. coli-encapsulated, recombinant modified peanut proteins Ara h 1, Ara h 2, and Ara h 3 (EMP-123) for the treatment of peanut allergy.
ABSTRACT: Immunotherapy for peanut allergy may be limited by the risk of adverse reactions.To investigate the safety and immunologic effects of a vaccine containing modified peanut proteins.This was a phase 1 trial of EMP-123, a rectally administered suspension of recombinant Ara h 1, Ara h 2, and Ara h 3, modified by amino acid substitutions at major IgE-binding epitopes, encapsulated in heat/phenol-killed E. coli. Five healthy adults were treated with 4 weekly escalating doses after which 10 peanut-allergic adults received weekly dose escalations over 10 weeks from 10 mcg to 3063 mcg, followed by three biweekly doses of 3063 mcg.There were no significant adverse effects in the healthy volunteers. Of the 10 peanut-allergic subjects [4 with intermittent asthma, median peanut IgE 33.3 kUA /l (7.2-120.2), and median peanut skin prick test wheal 11.3 mm (6.5-18)]; four experienced no symptoms; one had mild rectal symptoms; and the remaining five experienced adverse reactions preventing completion of dosing. Two were categorized as mild, but the remaining three were more severe, including one moderate reaction and two anaphylactic reactions. Baseline peanut IgE was significantly higher in the five reactive subjects (median 82.4 vs 17.2 kUA /l, P = 0.032), as was baseline anti-Ara h 2 IgE (43.3 versus 8.3, P = 0.036). Peanut skin test titration and basophil activation (at a single dilution) were significantly reduced after treatment, but no significant changes were detected for total IgE, peanut IgE, or peanut IgG4.Rectal administration of EMP-123 resulted in frequent adverse reactions, including severe allergic reactions in 20%.
Project description:Sublingual immunotherapy (SLIT) with peanut changes clinical and immune responses in most peanut-allergic individuals, but the response is highly variable.We sought to examine the component-specific effects of peanut SLIT and determine whether peanut component testing could predict the outcome of a double-blind, placebo-controlled food challenge (DBPCFC) after 12 months of peanut SLIT.We included 33 subjects who underwent peanut SLIT with a DBPCFC of 2500 mg of peanut protein performed after 12 months of therapy. Plasma samples from baseline and after 12 months of peanut SLIT were assayed using ImmunoCAP for IgE and IgG4 against whole peanut, Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9.Following 12 months of SLIT, 10 subjects (30%) passed the DBPCFC without symptoms and were considered desensitized. Subjects that failed the DBPCFC tolerated a median of 460 mg peanut protein (range: 10-1710 mg). The desensitized group had significantly lower baseline levels of IgE against peanut (median 40.8 vs. 231 kUA /L, P = 0.0082), Ara h 2 (median 17 vs. 113 kUA /L, P = 0.0082), and Ara h 3 (median 0.3 vs. 8.5 kUA /L, P = 0.0396). ROC curves indicated that baseline IgE against peanut and Ara h 2 were equally effective at discriminating between the two groups (AUC = 0.7957, P = 0.007752 for both).In this cohort of subjects undergoing SLIT for peanut allergy, lower baseline levels of IgE against Ara h 2, Ara h 3, and peanut were associated with successful desensitization.
Project description:Cross-linking of IgE antibody by specific epitopes on the surface of mast cells is a prerequisite for triggering symptoms of peanut allergy. IgE epitopes are frequently categorized as linear or conformational epitopes. Although linear IgE-binding epitopes of peanut allergens have been defined, little is known about conformational IgE-binding epitopes.To identify clinically relevant conformational IgE epitopes of the two most important peanut allergens, Ara h 2 and Ara h 6, using phage peptide library.A phage 12mer peptide library was screened with allergen-specific IgE from 4 peanut-allergic patients. Binding of the mimotopes to IgE from a total of 29 peanut-allergic subjects was measured by ELISA. The mimotope sequences were mapped on the surface areas of Ara h 2 and Ara h 6 using EpiSearch.Forty-one individual mimotopes were identified that specifically bind anti- Ara h 2/Ara h 6 IgE as well as rabbit anti-Ara h 2 and anti-Ara h 6 IgG. Sequence alignment showed that none of the mimotope sequences match a linear segment of the Ara h 2 or Ara h 6 sequences. EpiSearch analysis showed that all the mimotopes mapped to surface patches of Ara h 2 and Ara h 6. Eight of the mimotopes were recognized by more than 90% of the patients, suggesting immunodominance. Each patient had distinct IgE recognition patterns but the recognition frequency was not correlated to the concentration of peanut specific IgE or to clinical history.The mimotopes identified in this study represent conformational epitopes. Identification of similar surface patches on Ara h 2 and Ara h 6 further underscores the similarities between these two potent allergens.
Project description:Ara h 2 and Ara h 6 are moderately homologous and highly potent peanut allergens.To identify IgE-binding linear epitopes of Ara h 6, compare them to those of Ara h 2, and to stratify binding based on clinical histories.Thirty highly peanut-allergic subjects were stratified by clinical history. Sera were diluted to contain the same amount of anti-peanut IgE. IgE binding to overlapping 20-mer peptides of Ara h 2 and Ara h 6 was assessed using microarrays.Each subject had a unique IgE-binding fingerprint to peptides; these data were coalesced into epitope binding. IgE from subjects with a history of more severe reactions (n = 19) had a smaller frequency of binding events (BEs) for both Ara h 2 (52 BEs of 152 (19X8epitopes) possible BEs and Ara h 6 (13 BEs of 133 (19X7 epitopes) possible BEs) compared to IgE from those with milder histories (n = 11) (Ara h 2: 47 BEs of 88 (11X8 epitopes) possible BEs, P < 0.01; Ara h 6: 25 BEs of 77 (11X7 epitopes) possible BEs, P < 0.001). Using an unsupervised hierarchal cluster analysis, subjects with similar histories tended to cluster. We have tentatively identified a high-risk pattern of binding to peptides of Ara h 2 and Ara h 6, predominantly in subjects with a history of more severe reactions (OR = 12.6; 95% CI: 2.0-79.5; P < 0.01).IgE from patients with more severe clinical histories recognize fewer linear epitopes of Ara h 2 and Ara h 6 than do subjects with milder reactions and bind these epitopes in characteristic patterns. Close examination of IgE binding to epitopes of Ara h 2 and Ara h 6 may have prognostic value.
Project description:Peanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. Following our characterization of the two peanut allergens Ara h 1 and Ara h 2, we have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from approximately 45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3-allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions.
Project description:BACKGROUND:Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80-90% of peanut allergic patients. METHODS:The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients. RESULTS:The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP--Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct. DISCUSSION:The reduced IgE binding to the MBP--Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP--Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy.
Project description:Peanut allergy is an IgE-mediated adverse reaction to a subset of proteins found in peanuts. Immunotherapy aims to desensitize allergic patients through repeated and escalating exposures for several months to years using extracts or flours. The complex mix of proteins and variability between preparations complicates immunotherapy studies. Moreover, peanut immunotherapy is associated with frequent negative side effects and patients are often at risk of allergic reactions once immunotherapy is discontinued. Allergen-specific approaches using recombinant proteins are an attractive alternative because they allow more precise dosing and the opportunity to engineer proteins with improved safety profiles. We tested whether Ara h 1 and Ara h 2, two major peanut allergens, could be produced using chloroplast of the unicellular eukaryotic alga, Chlamydomonas reinhardtii. C. reinhardtii is novel host for producing allergens that is genetically tractable, inexpensive and easy to grow, and is able to produce more complex proteins than bacterial hosts. Compared to the native proteins, algal-produced Ara h 1 core domain and Ara h 2 have a reduced affinity for IgE from peanut-allergic patients. We further found that immunotherapy using algal-produced Ara h 1 core domain confers protection from peanut-induced anaphylaxis in a murine model of peanut allergy.
Project description:Peanut allergy continues to be a problem in most developed countries of the world. We sought a processing method that would alter allergenic peanut proteins, such that allergen recognition by IgE from allergic individuals would be significantly reduced or eliminated. Such a method would render accidental exposures to trace amounts of peanuts safer. A combination of boiling and frying decreased recovery of Ara h 1 and Ara h 2 at their expected MWs. In contrast, treatment with high pressures under varying temperatures had no effect on protein extraction profiles. Antibodies specific for Ara h 1, Ara h 2, and Ara h 6 bound proteins extracted from raw samples but not in boiled/fried samples. However, pre-incubation of serum with boiled/fried extract removed most raw peanut-reactive IgE from solution, including IgE directed to Ara h 1 and 2. Thus, this method of processing is unlikely to generate a peanut product tolerated by peanut allergic patients. Importantly, variability in individual patients' IgE repertoires may mean that some patients' IgE would bind fewer polypeptides in the sequentially processed seed.
Project description:Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens. One of the major peanut allergens, Ara h I, was selected from these clones using Ara h I specific oligonucleotides and polymerase chain reaction technology. The Ara h I clone identified a 2.3-kb mRNA species on a Northern blot containing peanut poly (A)+ RNA. DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants. The isolation of the Ara h I clones allowed the synthesis of this protein in E. coli cells and subsequent recognition of this recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity. With the production of the recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general
Project description:<h4>Background</h4>Peanut allergy is a life-threatening condition; there is currently no cure. While whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions and even fatalities in peanut allergy.<h4>Objective</h4>To identify short, HLA-degenerate CD4(+) T cell epitope-based peptides of the major peanut allergen Ara h 1 that target allergen-specific T cells without causing IgE-mediated inflammatory cell activation, as candidates for safe peanut-specific immunotherapy.<h4>Methods</h4>Ara h 1-specific CD4(+) T cell lines (TCL) were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using CFSE-based methodology. T cell epitopes were identified using CFSE and thymidine-based proliferation assays. Epitope HLA-restriction was investigated using blocking antibodies, HLA-genotyping and epitope prediction algorithms. Functional peanut-specific IgE reactivity to peptides was assessed by basophil activation assay.<h4>Results</h4>A total of 145 Ara h 1-specific TCL were generated from 18 HLA-diverse peanut-allergic subjects. The TCL recognized 20-mer peptides throughout Ara h 1. Nine 20-mers containing the most frequently recognized epitopes were selected and their recognition confirmed in 18 additional peanut-allergic subjects. Ten core epitopes were mapped within these 20-mers. These were HLA-DQ and/or HLA-DR restricted, with each presented on at least two different HLA-molecules. Seven short (? 20 aa) non-basophil-reactive peptides encompassing all core epitopes were designed and validated in peanut-allergic donor PBMC T cell assays.<h4>Conclusions and clinical relevance</h4>Short CD4(+) T cell epitope-based Ara h 1 peptides were identified as novel candidates for a safe, T cell targeted peanut-specific immunotherapy for HLA-diverse populations.
Project description:BACKGROUND:2S-albumins Ara h 2 and Ara h 6 are the most potent peanut allergens and levels of specific immunoglobulin E (IgE) towards these proteins are good predictors of clinical reactivity. Because of structural homologies, Ara h 6 is generally considered to cross-react extensively with Ara h 2. OBJECTIVE:We aimed to quantify the IgE cross-reactivity between Ara h 2 and Ara h 6. METHODS:Peanut 2S-albumins were purified from raw peanuts. The IgE cross-reactivity between Ara h 2 and Ara h 6 was evaluated with 32 sera from French and US peanut-allergic patients by measuring the residual IgE-binding to one 2S-albumin after depletion of IgE antibodies recognizing the other 2S-albumin. The IgE cross-reactivity between Ara h 2 and Ara h 6 was further investigated by competitive inhibition of IgE-binding and by a model of mast cell degranulation. RESULTS:A highly variable level of IgE cross-reactivity was revealed among the patients. The mean fraction of cross-reactive IgE antibodies represented only 17.1% of 2S-albumins-specific IgE antibodies and was lower than the mean fraction of IgE specific to Ara h 2 (57.4%) or to Ara h 6 (25.5%). The higher level of Ara h 2-specific IgE was principally due to the IgE-binding capacity of an insertion containing the repeated immunodominant linear epitope DPYSPOH S. The impact of IgE cross-reactivity on diagnostic testing was illustrated with a serum displaying an Ara h 6-specific IgE response of 26 UI/mL that was not associated with the capacity of Ara h 6 to trigger mast cell degranulation. CONCLUSIONS & CLINICAL RELEVANCE:Immunoglobulin E antibodies specific to peanut 2S-albumins are mainly non-cross-reactive, but low-affinity cross-reactivity can affect diagnostic accuracy. Testing IgE-binding to a mixture of 2S-albumins rather than to each separately may enhance diagnostic performance.