Original Articles: Environmental and Occupational Disorders
Mutational analysis of the IgE epitopes in the latex allergen Hev b 5,☆☆

https://doi.org/10.1067/mai.2001.115482Get rights and content

Abstract

Background: Hev b 5 is a major latex allergen and potential candidate for an immunotherapy reagent. Objective: The purpose of this study was to produce a hypoallergenic form of Hev b 5. Methods: We used SPOTs analysis with alanine substitution to identify amino acids (AAs) critical for IgE binding and used site-directed mutagenesis to produce recombinant proteins with altered IgE-binding activity. Results: Eleven epitopes were identified (5.1-5.11) in Hev b 5. Individual patients demonstrated variable epitope recognition, with the most intense reactivity to epitopes 5.4 and 5.7. IgE inhibition assays with synthetic peptides indicated that mutating a single epitope would not reduce IgE binding, but rather a combination of epitopes was required. After alanine substitutions to identify the important AAs, site-directed mutagenesis was used to replace the crucial AAs with alanine. Twenty clones with different combinations of altered epitopes were evaluated by means of IgE inhibition assays. Clones with mutations in single epitopes failed to reduce IgE binding, but changes to 8 epitopes (14 AAs) resulted in a 4500-fold reduction in IgE binding. Epitopes 5.7 and 5.9 were found to be cross-reactive, making Hev b 5 a multivalent allergen. Conclusions: We produced a recombinant Hev b 5 protein with significantly reduced IgE-binding activity. Changing a minimum of 3 immunodominant epitopes was required to cause a 100-fold reduction in IgE binding. Changes in 8 epitopes, particularly the cross-reactive epitopes 5.7 and 5.9, were needed to maximize the reduction in IgE binding. Mutants with reduced IgE-binding activity may prove to be valuable reagents for immunotherapy. (J Allergy Clin Immunol 2001;107:1069-76.)

Section snippets

Antibodies

Human sera with anti-Hev b 5 IgE were obtained from 7 health-care workers diagnosed with latex allergy who had positive skin test responses to recombinant (r) Hev b 5 and demonstrated skin test reactivity at 1 ng/mL or less.15 Rabbit anti-Hev b 5 IgG was obtained from a New Zealand white rabbit immunized with 200 μg of maltose-binding protein/rHev b 5 in a 50:50 mixture of TiterMax adjuvant (CytRx, Norcross, Ga). The rabbit was given a booster injection at 3 months and was bled 4 weeks later,

Immunodominant Hev b 5 epitopes

We have previously reported 6 epitopes for human IgE (epitopes 1, 2, 4, 6, 7, and 10) that were identified by using SPOTs analysis with sera pooled from 10 health-care workers with latex allergy.17 An additional IgE-binding epitope (epitope 8) was detected by using sera from an individual patient with latex allergy (unpublished data). We now examine the Hev b 5 sequence by using sera from a second group of 7 patients selected because they had a positive skin prick test response to 1 ng/mL or

Discussion

Patients with latex allergy are often hyperreactive to latex protein, making immunotherapy risky. We have shown that recombinant latex proteins are an important source of purified latex allergens that may serve to improve in vitro and in vivo diagnostic tests.15 The production of recombinant proteins and the potential to modify the cDNA for these proteins allow novel approaches for immunotherapy. It has been proposed that site-directed mutagenesis be used to produce hypoallergenic variants of

Acknowledgements

We thank Maren Nalepa, Colleen Landy, Ben McClintic, and Deidre Blake for excellent technical assistance.

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Supported by the LEAP Testing Service of the Guthrie Research Institute.

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Reprint requests: Donald Beezhold, PhD, Director, Laboratory of Immunobiology, Guthrie Research Institute, 1 Guthrie Square, Sayre, PA 18840.

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