Modification of Peanut Allergen Ara h 3: Effects on IgE Binding and T Cell Stimulation

Rabjohn, Pat; West, Charles; Connaughton, Cathie; Sampson, Hugh A.; Helm, Ricki M.; Burks, A. Wesley; Bannon, Gary A.

doi:10.1159/000057999

Cited by 78 publications

(57 citation statements)

References 16 publications

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“…[27, 28]]. But again, in contrast to our study, often more than one amino acid per linear epitope was exchanged, and in most cases, many different amino acids were mutated in each molecule.…”

Section: Discussioncontrasting

confidence: 59%

Lysine as a Critical Amino Acid for IgE Binding in Phl p 5b C Terminus

Gehlhar

Rajashankar²,

Hofmann³

et al. 2006

Int Arch Allergy Immunol

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Background: Allergens induce the formation of specific immunoglobulin (Ig)E and harbor at least two IgE-binding regions (epitopes) to facilitate crosslinking of basophilic or mast-cell-bound specific IgE antibodies. Studies mapping linear epitopes have shown that these regions often contain charged or hydrophobic amino acids. Nevertheless, these studies are hampered by limited significance due to the often conformational nature of IgE epitopes. This prompted us to study the role of lysines in the context of an intact 3-dimensional model. Methods: Major allergen Phl p 5b from timothy grass bears 12 lysines in its C-terminal half. Using site-directed mutagenesis, we substituted all 10 surface-exposed lysines by alanines. Results: Although structural integrity of the lysine-deficient mutant was not altered, IgE-binding capacity measured by ELISA inhibition tests and crosslinking activity in ex vivo basophil stimulation and in vivo skin prick tests were significantly diminished. Interestingly, binding of specific IgG antibodies was considerably less reduced by loss of lysines. Conclusion: Lysine is an important amino acid for IgE binding in more than one epitope of major grass pollen allergen Phl p 5b C terminus. Allergenicity, but not IgG binding of the molecule, is substantially diminished by single amino acid substitutions without structural integrity being hampered.

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Section: Discussioncontrasting

confidence: 59%

Lysine as a Critical Amino Acid for IgE Binding in Phl p 5b C Terminus

Gehlhar

Rajashankar²,

Hofmann³

et al. 2006

Int Arch Allergy Immunol

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“…Similar attributes have been engineered into peanut allergen Ara h 3 [32]. Since there are three major peanut allergens (Ara h 1, Ara h 2, and Ara h 3) [33], it seems likely that it will be possible to produce hypoallergenic forms of all three allergens, and, when coupled with an appropriate adjuvant, be a potentially safe, effective treatment for peanut allergy.…”

Section: Discussionmentioning

confidence: 99%

Allergenic characteristics of a modified peanut allergen

King

Helm

Stanley

et al. 2005

Mol. Nutr. Food Res.

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Attempts to treat peanut allergy using traditional methods of allergen desensitization are accompanied by a high risk of anaphylaxis. The aim of this study was to determine if modifications to the IgE-binding epitopes of a major peanut allergen would result in a safer immunotherapeutic agent for the treatment of peanut-allergic patients. IgE-binding epitopes on the Ara h 2 allergen were modified, and modified Ara h 2 (mAra h 2) protein was produced. Wild-type (wAra h 2) and mAra h 2 proteins were analyzed for their ability to interact with T-cells, their ability to bind IgE, and their ability to release mediators from a passively sensitized RBL-2H3 cell line. Multiple T-cell epitopes were identified on the major peanut allergen, Ara h 2. Ara h 2 amino acid regions 11-35, 86-125, and 121-155 contained the majority of peptides that interact with T-cells from most patients. The wAra h 2 and mAra h 2 proteins stimulated proliferation of T-cells from peanut-allergic patients to similar levels. In contrast, the mAra h 2 protein exhibited greatly reduced IgE-binding capacity compared to the wild-type allergen. In addition, the modified allergen released significantly lower amounts of beta-hexosaminidase, a marker for IgE-mediated RBL-2H3 degranulation, compared to the wild-type allergen.

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“…This was performed for the peanut allergens Ara h 1, 2, and 3. Twenty three IgE-binding regions were found for the 63kDa Ara h 1 [23,24], 10 for the 17 kDa Ara h 2 [25], and only four for the 50 kDa Ara h 3 [26]. It was found that single amino acid substitutions in the peptides could remove most IgE-binding activity [23,24].…”

Section: Engineering Out Ige-binding Sequencesmentioning

confidence: 97%

Genetically engineered vaccines

Thomas

Hales

Smith

2005

Curr Allergy Asthma Rep

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The application of recombinant DNA technology to allergen research has provided the sequence information and genetic material to produce new types of allergy vaccines. One general strategy has been to use the knowledge to produce synthetic peptides that represent selected T-cell or B-cell epitopes. The production of genetically engineered allergens provides an alternative strategy to construct hypoallergenic vaccines, which can provide a better and less selected representation of the epitopes. Many strategies have been used to produce such hypoallergens, and their ability to reduce allergenicity has been amply demonstrated by skin and nasal provocation tests. The retention of T cell-stimulating activity has also been demonstrated, and a consistent feature of the vaccines has been, despite the reduced immunoglobulin E (IgE)-binding reactivity, the ability to induce anti-allergen IgG antibody. The lead hypoallergens have been polypeptide fragments and trimeric constructs of the birch allergen Bet v 1. A clinical trial with these medicaments has shown the ability to modify IgE and IgG antibody production, skin test reactivity, and symptom scores. This is the first trial of a recombinant allergy vaccine, and it has set a benchmark for further studies. A new generation of hypoallergens is now being produced based on the detailed knowledge of the tertiary structures of the allergens and of the T-cell and B-cell epitopes. The modifications have been made to change the topography of the allergens while retaining a stable, folding structure. In the case of Bet v 1, tertiary structures of hypoallergens have been determined. Structurally modeled hypoallergens have been produced for pollen, venom, food, and latex allergens, with promising characteristics from preclinical studies.

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Modification of Peanut Allergen Ara h 3: Effects on IgE Binding and T Cell Stimulation

Cited by 78 publications

References 16 publications

Lysine as a Critical Amino Acid for IgE Binding in Phl p 5b C Terminus

Lysine as a Critical Amino Acid for IgE Binding in Phl p 5b C Terminus

Allergenic characteristics of a modified peanut allergen

Genetically engineered vaccines

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