- Email: [email protected]
714 Evidence of No cross-reactivity between the Hevea brasiliensis latex algebra Hev b 7 and potato patatins and proteins from banana and avocado
Abstracts
J ALLERGY CLIN IMMUNOL VOLUME 104. NUMBER 1. PART 2
712
713
Site-directed Mutagenesis of the Latex Allergen Hev b 5 E./ Paupore. JE Slarer Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD Hev b 5 has been identified as an important allergen in natural rubber latex and may be a target for use in peptide immunotherapy. We (Mol. 1mmuno1.1999;35:135) and Beezhold et al. (JACI 1999;103: 1166) have identified several potential murine. rabbit and human B-cell epitopes of Hev b 5, with KEXE and KXEE as common motifs in the antibody binding sites. In this study we used sitedirected mutagenesis by a PCR method to introduce a lysine to proline mutation in the KTEE sequence at position 28 of Hev b 5. PCR-based site-directed mutagenesis with a megaprimer intermediate was performed according to the one-tube protocol described by Marini et al. (Nucleic Acids Research 1993; 21:2277). The megaprimer was generated with Hev b 5IpMAL as the template, left primer 5’- CGGAATTCTCCTTCATTTTTGCTTTCCAA-3’. and right primer 5’- CAGGTTCTTCGGT*TGG*CGTC3’, with the mutant site marked with asterisks. The first round PCR conditions were: 40 cycles, 2 min at 94°C. I min at 50°C. I min at 72°C total volume 50 pL. In a second PCR step, we used 50 pL of the megaprimer per reaction tube along with a new right primer 5’CGTCGACTTACTTCTCGGTITCAGGAGCTGG-3’ and the following conditions: 40 cycles, 2 min at 94”C, I min at 52°C. I min at 72°C. total volume 100 l.tL. To minimize terminal transferase activity, all reactions were run with Vent R polymerase. The final product was then digested and ligated into the pMAL-c2 expression vector (New England Biolabs). The ligation products were digested with restriction enzymes to confirm the insertion sequence size, and were sequenced by terminal dideoxynucleotide reactions that were analyzed on an A91 Prism Model 377 automated sequencer. We then transformed XL-I Blue cells with the ligated plasmids. After induction with IPTG, the maltose binding protein-fusion proteins were purified on an amylose resin and analyzed by SDS-PAGE. Two clones were identified that contained an insertion sequence of appropriate size (229 bp). DNA sequencing confirmed the presence of the AAG to CCA mutation at positions 170-172 in both clones. This confirmed a lysine to proline replacement in position 28 of the Hev b 5 polypeptide. On the SDS-PAGE gel, the induced sample displayed a single band at 66 kDa. This is consistent with the in-frame expression of the mutant insert sequence. We have successfully generated and expressed a Hev b 5 fusion protein containing a mutation in one of the identified antibodybinding sites. This mutant, and others, will be used to further characterize the B-cell epitopes of Hev b 5. Human T Cell Epitopes of the Latex Allergen Hev b 5 in Health Care Workers Hnrini de Silva*. Michael Sutherland*. Cenk Suphioglu *, Sue McLellan *, Jay Slaterf#$12, Jenny Rolland*, Robyn O’Hehir* *Department of Allergy, Asthma and Clinical Immunology. Monash University Medical School, The Alfred Hospital, Prahran. Victoria, Australia tLaboratory of Immunobiochemistry, Center for Biologics Evaluation and Research, US Food and Drug Administration, Rockville. MD BACKGROUND: Latex allergy is a potentially severe, untreatable allergic disease affecting health care workers as a high risk cohort. Hev b 5 is a major latex allergen reacting with serum IgE from 92% of latex allergic health care workers. As CD4+ T cell recognition is central to the specific immune response to allergens, identification of dominant T cell epitopes is critical for the development of specific immunotherapy for latex allergy. AIM: Identification of T cell epitopes of Hev b 5 in health care workers.
S241
METHODS: Six latex allergic health care workers (grade 34 EAST score) were studied. Peripheral blood latex specific threeweek T cell lines were generated and screened for proliferative response to overlapping 20-mer peptides of Hev b 5. Supematants collected at 48 hours were analysed by ELISA for IL-5 and IFNy. RESULTS: Dot immunoblotting using recombinant Hev b S/MBP fusion protein indicated serum specific IgE in five of six patients. T cell reactivity to one or more Hev b 5 peptides was identified in these five donors, but not in the sixth. Hev b 5(46-65) induced T cell proliferation in all five donors. Hev b 5(109-128) stimulated T cells from three of these patients. Proliferative responses were accompanied by substantial IL-5 secretion and minimal IFNy indicating a ThZpredominant cytokine profile. CONCLUSIONS: Five of six latex allergic patients demonstrated T cell responsiveness to Hev b 5 consistent with a major T cell reactive latex allergen. no T cell immunodominant regions of Hev b 5 were identified and reactivity to these sites was associated with strong IL-5 but minimal IFNy production. Financial support from the National Health and Medical Research Council and the Australian Allergy Foundation is acknowledged. 714
Evidence of No Cross-reactivity Between the Hevea Brasiliensls Latex Allergen Hev b 7 and Potato Patatins and Proteins from Banana and Avocado Christine Hafner; Slawomir Sowka. Orro Scheiner; Don Beezhold, Heimo Breiteneder Evidence of no Cross-reactivity Between the Hevea brasiliensis Latex Allergen Hev b 7 and Potato Patatin and Proteins from Banana and Avocado C. Hafner+, S. Sowka+, 0. Scheiner+, D. Beezhold*) and H. Breiteneder+, Dept. of General and Experimental Pathology, University of Vienna, Medical School, Austria+; Guthrie Research Institute, Sayre, PA, USA*) Since the first clinical description of latex allergy, IgE-mediated hypersensitivity to natural rubber latex proteins has been known to impose serious medical problems, especially among occupationally exposed persons and patients who have to undergo repeated surgeries such as children with spina bitida. Hev b 7 is a Hevea brasiliensis latex allergen with sequence identities of 39-42% to patatins recently identified as potato allergens. The aim of this study was to evaluate possible cross-reactivities between Hev b 7, patatins and proteins from banana and avocado, and to compare the IgE reactivity of four Hev b 7 isoforms in vitro and in viva. A Hevea brasiliensis IZAP cDNA library was screened using a Hev b 7 cDNA probe. Four Hev b 7 isofotms were identified and produced in recombinant form in the methylotrophic yeast Pichia pastoris. IgE immunoblot inhibitions and ELISA inhibition assays were used to investigate possible cross-reactivities between recombinant Hev b 7 and potato patatin and proteins from avocado and banana. All four recombinant isoforms displayed identical IgE binding capacities in IgE immunoblots. In addition these isoforms were evaluated in skin prick tests and provoked responses equivalent to natural Hev b 7. No inhibition was observed between Hev b 7 isoforms and potato patatins and proteins from avocado and banana in ELISA inhibition and IgE immunoblot inhibition experiments. Therefore, all four recombinant Hev b 7 isoforms. potato patatins and their homologues appear not to contribute to cross-reactivities in the latex-fruit syndrome. This work was in part supported by the Austrian Science Fund grant Pl2838-GEN
J ALLERGY CLIN IMMUNOL VOLUME 104. NUMBER 1. PART 2
712
713
Site-directed Mutagenesis of the Latex Allergen Hev b 5 E./ Paupore. JE Slarer Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD Hev b 5 has been identified as an important allergen in natural rubber latex and may be a target for use in peptide immunotherapy. We (Mol. 1mmuno1.1999;35:135) and Beezhold et al. (JACI 1999;103: 1166) have identified several potential murine. rabbit and human B-cell epitopes of Hev b 5, with KEXE and KXEE as common motifs in the antibody binding sites. In this study we used sitedirected mutagenesis by a PCR method to introduce a lysine to proline mutation in the KTEE sequence at position 28 of Hev b 5. PCR-based site-directed mutagenesis with a megaprimer intermediate was performed according to the one-tube protocol described by Marini et al. (Nucleic Acids Research 1993; 21:2277). The megaprimer was generated with Hev b 5IpMAL as the template, left primer 5’- CGGAATTCTCCTTCATTTTTGCTTTCCAA-3’. and right primer 5’- CAGGTTCTTCGGT*TGG*CGTC3’, with the mutant site marked with asterisks. The first round PCR conditions were: 40 cycles, 2 min at 94°C. I min at 50°C. I min at 72°C total volume 50 pL. In a second PCR step, we used 50 pL of the megaprimer per reaction tube along with a new right primer 5’CGTCGACTTACTTCTCGGTITCAGGAGCTGG-3’ and the following conditions: 40 cycles, 2 min at 94”C, I min at 52°C. I min at 72°C. total volume 100 l.tL. To minimize terminal transferase activity, all reactions were run with Vent R polymerase. The final product was then digested and ligated into the pMAL-c2 expression vector (New England Biolabs). The ligation products were digested with restriction enzymes to confirm the insertion sequence size, and were sequenced by terminal dideoxynucleotide reactions that were analyzed on an A91 Prism Model 377 automated sequencer. We then transformed XL-I Blue cells with the ligated plasmids. After induction with IPTG, the maltose binding protein-fusion proteins were purified on an amylose resin and analyzed by SDS-PAGE. Two clones were identified that contained an insertion sequence of appropriate size (229 bp). DNA sequencing confirmed the presence of the AAG to CCA mutation at positions 170-172 in both clones. This confirmed a lysine to proline replacement in position 28 of the Hev b 5 polypeptide. On the SDS-PAGE gel, the induced sample displayed a single band at 66 kDa. This is consistent with the in-frame expression of the mutant insert sequence. We have successfully generated and expressed a Hev b 5 fusion protein containing a mutation in one of the identified antibodybinding sites. This mutant, and others, will be used to further characterize the B-cell epitopes of Hev b 5. Human T Cell Epitopes of the Latex Allergen Hev b 5 in Health Care Workers Hnrini de Silva*. Michael Sutherland*. Cenk Suphioglu *, Sue McLellan *, Jay Slaterf#$12, Jenny Rolland*, Robyn O’Hehir* *Department of Allergy, Asthma and Clinical Immunology. Monash University Medical School, The Alfred Hospital, Prahran. Victoria, Australia tLaboratory of Immunobiochemistry, Center for Biologics Evaluation and Research, US Food and Drug Administration, Rockville. MD BACKGROUND: Latex allergy is a potentially severe, untreatable allergic disease affecting health care workers as a high risk cohort. Hev b 5 is a major latex allergen reacting with serum IgE from 92% of latex allergic health care workers. As CD4+ T cell recognition is central to the specific immune response to allergens, identification of dominant T cell epitopes is critical for the development of specific immunotherapy for latex allergy. AIM: Identification of T cell epitopes of Hev b 5 in health care workers.
S241
METHODS: Six latex allergic health care workers (grade 34 EAST score) were studied. Peripheral blood latex specific threeweek T cell lines were generated and screened for proliferative response to overlapping 20-mer peptides of Hev b 5. Supematants collected at 48 hours were analysed by ELISA for IL-5 and IFNy. RESULTS: Dot immunoblotting using recombinant Hev b S/MBP fusion protein indicated serum specific IgE in five of six patients. T cell reactivity to one or more Hev b 5 peptides was identified in these five donors, but not in the sixth. Hev b 5(46-65) induced T cell proliferation in all five donors. Hev b 5(109-128) stimulated T cells from three of these patients. Proliferative responses were accompanied by substantial IL-5 secretion and minimal IFNy indicating a ThZpredominant cytokine profile. CONCLUSIONS: Five of six latex allergic patients demonstrated T cell responsiveness to Hev b 5 consistent with a major T cell reactive latex allergen. no T cell immunodominant regions of Hev b 5 were identified and reactivity to these sites was associated with strong IL-5 but minimal IFNy production. Financial support from the National Health and Medical Research Council and the Australian Allergy Foundation is acknowledged. 714
Evidence of No Cross-reactivity Between the Hevea Brasiliensls Latex Allergen Hev b 7 and Potato Patatins and Proteins from Banana and Avocado Christine Hafner; Slawomir Sowka. Orro Scheiner; Don Beezhold, Heimo Breiteneder Evidence of no Cross-reactivity Between the Hevea brasiliensis Latex Allergen Hev b 7 and Potato Patatin and Proteins from Banana and Avocado C. Hafner+, S. Sowka+, 0. Scheiner+, D. Beezhold*) and H. Breiteneder+, Dept. of General and Experimental Pathology, University of Vienna, Medical School, Austria+; Guthrie Research Institute, Sayre, PA, USA*) Since the first clinical description of latex allergy, IgE-mediated hypersensitivity to natural rubber latex proteins has been known to impose serious medical problems, especially among occupationally exposed persons and patients who have to undergo repeated surgeries such as children with spina bitida. Hev b 7 is a Hevea brasiliensis latex allergen with sequence identities of 39-42% to patatins recently identified as potato allergens. The aim of this study was to evaluate possible cross-reactivities between Hev b 7, patatins and proteins from banana and avocado, and to compare the IgE reactivity of four Hev b 7 isoforms in vitro and in viva. A Hevea brasiliensis IZAP cDNA library was screened using a Hev b 7 cDNA probe. Four Hev b 7 isofotms were identified and produced in recombinant form in the methylotrophic yeast Pichia pastoris. IgE immunoblot inhibitions and ELISA inhibition assays were used to investigate possible cross-reactivities between recombinant Hev b 7 and potato patatin and proteins from avocado and banana. All four recombinant isoforms displayed identical IgE binding capacities in IgE immunoblots. In addition these isoforms were evaluated in skin prick tests and provoked responses equivalent to natural Hev b 7. No inhibition was observed between Hev b 7 isoforms and potato patatins and proteins from avocado and banana in ELISA inhibition and IgE immunoblot inhibition experiments. Therefore, all four recombinant Hev b 7 isoforms. potato patatins and their homologues appear not to contribute to cross-reactivities in the latex-fruit syndrome. This work was in part supported by the Austrian Science Fund grant Pl2838-GEN