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Crystal Structures of Der f 1, Der p 1 and a Der f 1 Allergen-Antibody Complex: Localization of Antibody Binding Epitopes
Abstracts S209
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Autoimmune Neutropenia Presenting with Severe, Lifethreatening Infections A. I. Gheller-Rigoni1, T. R. Adamiak1, B. R. Curtis2, M. K. Hintermeyer3, W. J. Grossman1; 1Medical College of Wisconsin, Milwaukee, WI, 2 BloodCenter of Wisconsin, Milwaukee, WI, 3Children’s Hospital of Wisconsin, Milwaukee, WI. RATIONALE: Autoimmune neutropenia (AIN) is caused by antineutrophil antibodies directed against neutrophil-specific antigens that cause neutrophil depletion and/or inhibition of function. Prior reports indicate that only 12% of patients present with serious infections. Here we characterize patients with AIN evaluated at our institution from 2006 to 2008. METHODS: Thirty patients, ages 0 to 19 years old, with confirmed antineutrophil antibodies, had their charts analyzed for the following parameters at the time of diagnosis: age, gender, nadir ANC, type of infections, presence of HLA antibodies, other autoimmune cytopenias, quantitative immunoglobulins, lymphocyte enumeration with subset analysis and treatment. RESULTS: Age at diagnosis ranged from 3.5 months to 18.5 years (mean 5 54 months) with a male to female distribution of 1.3:1. Eleven (37%) of the patients did not have neutropenia at presentation and 23 (77%) presented with severe infections requiring hospitalization. There was no difference in nadir ANCs between hospitalized and non-hospitalized groups. Four (13%) patients presented with recurrent perianal fistulas and/or abscesses and six (20%) had culture negative, recurrent oral ulcers. Ten (33%) of the patients had refractory autoimmune cytopenias and 19 (73%) had other abnormal immune studies (n 5 26). A number of the patients required treatment with GCSF and/or immunomodulators (IVIG/steroids/rituximab), resulting in resolution of infection. CONCLUSIONS: Contrary to traditional beliefs, AIN can present with life-threatening infections. In this cohort, 77% presented with severe infections requiring hospitalization. AIN should be considered in children who present with recurrent or serious infections, regardless of neutrophil count.
804
Crystal Structures of Der f 1, Der p 1 and a Der f 1 AllergenAntibody Complex: Localization of Antibody Binding Epitopes M. Chruszcz1, M. D. Chapman2, E. A. Stura3, L. D. Vailes2, J. SaintRemy4, W. Minor1, A. Pome´s2; 1University of Virginia, Charlottesville, VA, 2INDOOR Biotechnologies, Inc., Charlottesville, VA, 3Commissariat a` l’Energie Atomique Saclay, Gif-sur-Yvette, France, 4University of Leuven, Leuven, Belgium. RATIONALE: The cysteine mite proteases Der p 1 and Der f 1 show 82% amino acid sequence identity and are immunodominant mite allergens. Human IgE antibody responses to Group 1 allergens are strongly cross-reactive, whereas murine IgG antibody responses are largely species specific. To analyze the antigenic surface of these allergens, we elucidated the crystal structures of Der f 1, Der p 1 and the complex of Der f 1 with a crossreacting monoclonal antibody (4C1). ˚ , 1.4A ˚ METHODS: Three molecular structures were determined at 2.0A ˚ resolution, respectively, using the X-ray diffraction method and 1.9A from crystals of natural Der f 1, recombinant Der p 1 and Fab fragments of 4C1. RESULTS: Structural comparison of Der f 1 and Der p 1 revealed that surfaces near the catalytic cysteine-35/34 were almost identical, whereas four major superficial patches differed significantly. Calcium, instead of the originally described magnesium, was bound to rDer p 1. A model of the Der f 1-4C1 complex, showed that the cross-reactive antibody binds to amino acids in common between both allergens, in a region of mostly non-conserved residues. DNA encoding for Der f 1 has been mutated for expression of single epitope mutants in Pichia pastoris to analyze the effect of specific amino acids on IgE antibody binding. CONCLUSIONS: Analysis of high resolution structures of Group 1 allergens in complex with antibodies revealed antigenic determinants involved in cross-reactivity. Identification of allergenic epitopes will significantly influence the development of new recombinant vaccines for the treatment of mite allergic disease.
805
Design of Hypoallergenic Derivatives of Cyn d 1, a Major Allergen from Bermuda grass pollen R. Tiwari1, P. L. Bhalla2, M. B. Singh2; 1Massey University, Palmerston North 4442, New Zealand, 2The University of Melbourne, Faculty of Land and Food Resources, Melbourne, VIC 3052, Australia. RATIONALE: Group 1 grass pollen allergens, such as Cyn d 1 are members of the plant expansin family, which have been shown to be structurally related. The deduced amino acid sequences of the b-expansin family of proteins and group 1 grass pollen allergens show an overall sequence identity of between 40-45%. We expressed C-terminal half of Arabidopsis EXPB1 that is homologous to the IgE epitope-containing region of rCyn d 1 and found that this segment did not react with serum IgE from BGPallergic patients. The aim was to alter critical residues to disrupt the IgE epitopes of Cyn d 1 and make it more like the non-allergenic EXPB1. METHODS: To select residues from Cyn d 1 that are critical for IgE binding, the Cyn d 1 epitopes were scanned by site-directed mutagenesis taking into account the sequence of homologous non-allergenic b-expansin gene (EXPB1) from Arabidopsis. Target amino acid substitutions were selected using sequence information from the analogous sites in Cyn d 1 and EXPB1. Nine potential IgE-interacting residues were identified. Based on these residues, eight mutant Cyn d 1 variants with combinations of mutated residues were produced and expressed. RESULTS: Immuno dot blot and ELISAs indicated that three out of eight mutants showed significantly reduced IgE reactivity, compared to WT rCyn d 1. CONCLUSIONS: This approach of using structurally homologous nonallergenic proteins as models to identify critical IgE binding residues in the allergenic counterpart may be generally applied to produce variants of those allergenic proteins whose detailed 3-D structures are not yet available.
806
Carbohydrate Moieties Mediate Allergen Uptake by Human Dendritic Cells A. M. Ghaemmaghami, M. Emara, F. Shakib; The University of Nottingham, Nottingham, United Kingdom. RATIONALE: Dendritic cells (DC) play a pivotal role in the initiation and maintenance of allergic responses. In this study we aimed to elucidate the mechanisms of allergen uptake by human DC. METHODS: A combination of gene silencing (siRNA), blocking experiments and flowcytometry were used to investigate the uptake of a diverse range of clinically relevant allergens (Der p 1, Can f 1, Ara h 1 and Fel d 1) by human DC. We also quantified the carbohydrate content of allergens and used a number of deglycosylated allergen preparations to determine the role of sugars in allergen uptake by DC. RESULTS: Our data shows that the mannose receptor, a C type lectin expressed by DC, acts as a major receptor for a number of allergens from diverse sources and that the level of allergen uptake is directly linked to the degree of the allergen glycosylation. We have also shown that down-regulation of the mannose receptor or the use of deglycosylated allergens leads to a significant reduction in allergen uptake (n 5 5, P < 0.05) by DC. CONCLUSIONS: The mannose receptor appears to be involved in the uptake of different allergens by human DC, and that the glycosylation state of the allergen is likely to play an important role in this process.
MONDAY
803
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Autoimmune Neutropenia Presenting with Severe, Lifethreatening Infections A. I. Gheller-Rigoni1, T. R. Adamiak1, B. R. Curtis2, M. K. Hintermeyer3, W. J. Grossman1; 1Medical College of Wisconsin, Milwaukee, WI, 2 BloodCenter of Wisconsin, Milwaukee, WI, 3Children’s Hospital of Wisconsin, Milwaukee, WI. RATIONALE: Autoimmune neutropenia (AIN) is caused by antineutrophil antibodies directed against neutrophil-specific antigens that cause neutrophil depletion and/or inhibition of function. Prior reports indicate that only 12% of patients present with serious infections. Here we characterize patients with AIN evaluated at our institution from 2006 to 2008. METHODS: Thirty patients, ages 0 to 19 years old, with confirmed antineutrophil antibodies, had their charts analyzed for the following parameters at the time of diagnosis: age, gender, nadir ANC, type of infections, presence of HLA antibodies, other autoimmune cytopenias, quantitative immunoglobulins, lymphocyte enumeration with subset analysis and treatment. RESULTS: Age at diagnosis ranged from 3.5 months to 18.5 years (mean 5 54 months) with a male to female distribution of 1.3:1. Eleven (37%) of the patients did not have neutropenia at presentation and 23 (77%) presented with severe infections requiring hospitalization. There was no difference in nadir ANCs between hospitalized and non-hospitalized groups. Four (13%) patients presented with recurrent perianal fistulas and/or abscesses and six (20%) had culture negative, recurrent oral ulcers. Ten (33%) of the patients had refractory autoimmune cytopenias and 19 (73%) had other abnormal immune studies (n 5 26). A number of the patients required treatment with GCSF and/or immunomodulators (IVIG/steroids/rituximab), resulting in resolution of infection. CONCLUSIONS: Contrary to traditional beliefs, AIN can present with life-threatening infections. In this cohort, 77% presented with severe infections requiring hospitalization. AIN should be considered in children who present with recurrent or serious infections, regardless of neutrophil count.
804
Crystal Structures of Der f 1, Der p 1 and a Der f 1 AllergenAntibody Complex: Localization of Antibody Binding Epitopes M. Chruszcz1, M. D. Chapman2, E. A. Stura3, L. D. Vailes2, J. SaintRemy4, W. Minor1, A. Pome´s2; 1University of Virginia, Charlottesville, VA, 2INDOOR Biotechnologies, Inc., Charlottesville, VA, 3Commissariat a` l’Energie Atomique Saclay, Gif-sur-Yvette, France, 4University of Leuven, Leuven, Belgium. RATIONALE: The cysteine mite proteases Der p 1 and Der f 1 show 82% amino acid sequence identity and are immunodominant mite allergens. Human IgE antibody responses to Group 1 allergens are strongly cross-reactive, whereas murine IgG antibody responses are largely species specific. To analyze the antigenic surface of these allergens, we elucidated the crystal structures of Der f 1, Der p 1 and the complex of Der f 1 with a crossreacting monoclonal antibody (4C1). ˚ , 1.4A ˚ METHODS: Three molecular structures were determined at 2.0A ˚ resolution, respectively, using the X-ray diffraction method and 1.9A from crystals of natural Der f 1, recombinant Der p 1 and Fab fragments of 4C1. RESULTS: Structural comparison of Der f 1 and Der p 1 revealed that surfaces near the catalytic cysteine-35/34 were almost identical, whereas four major superficial patches differed significantly. Calcium, instead of the originally described magnesium, was bound to rDer p 1. A model of the Der f 1-4C1 complex, showed that the cross-reactive antibody binds to amino acids in common between both allergens, in a region of mostly non-conserved residues. DNA encoding for Der f 1 has been mutated for expression of single epitope mutants in Pichia pastoris to analyze the effect of specific amino acids on IgE antibody binding. CONCLUSIONS: Analysis of high resolution structures of Group 1 allergens in complex with antibodies revealed antigenic determinants involved in cross-reactivity. Identification of allergenic epitopes will significantly influence the development of new recombinant vaccines for the treatment of mite allergic disease.
805
Design of Hypoallergenic Derivatives of Cyn d 1, a Major Allergen from Bermuda grass pollen R. Tiwari1, P. L. Bhalla2, M. B. Singh2; 1Massey University, Palmerston North 4442, New Zealand, 2The University of Melbourne, Faculty of Land and Food Resources, Melbourne, VIC 3052, Australia. RATIONALE: Group 1 grass pollen allergens, such as Cyn d 1 are members of the plant expansin family, which have been shown to be structurally related. The deduced amino acid sequences of the b-expansin family of proteins and group 1 grass pollen allergens show an overall sequence identity of between 40-45%. We expressed C-terminal half of Arabidopsis EXPB1 that is homologous to the IgE epitope-containing region of rCyn d 1 and found that this segment did not react with serum IgE from BGPallergic patients. The aim was to alter critical residues to disrupt the IgE epitopes of Cyn d 1 and make it more like the non-allergenic EXPB1. METHODS: To select residues from Cyn d 1 that are critical for IgE binding, the Cyn d 1 epitopes were scanned by site-directed mutagenesis taking into account the sequence of homologous non-allergenic b-expansin gene (EXPB1) from Arabidopsis. Target amino acid substitutions were selected using sequence information from the analogous sites in Cyn d 1 and EXPB1. Nine potential IgE-interacting residues were identified. Based on these residues, eight mutant Cyn d 1 variants with combinations of mutated residues were produced and expressed. RESULTS: Immuno dot blot and ELISAs indicated that three out of eight mutants showed significantly reduced IgE reactivity, compared to WT rCyn d 1. CONCLUSIONS: This approach of using structurally homologous nonallergenic proteins as models to identify critical IgE binding residues in the allergenic counterpart may be generally applied to produce variants of those allergenic proteins whose detailed 3-D structures are not yet available.
806
Carbohydrate Moieties Mediate Allergen Uptake by Human Dendritic Cells A. M. Ghaemmaghami, M. Emara, F. Shakib; The University of Nottingham, Nottingham, United Kingdom. RATIONALE: Dendritic cells (DC) play a pivotal role in the initiation and maintenance of allergic responses. In this study we aimed to elucidate the mechanisms of allergen uptake by human DC. METHODS: A combination of gene silencing (siRNA), blocking experiments and flowcytometry were used to investigate the uptake of a diverse range of clinically relevant allergens (Der p 1, Can f 1, Ara h 1 and Fel d 1) by human DC. We also quantified the carbohydrate content of allergens and used a number of deglycosylated allergen preparations to determine the role of sugars in allergen uptake by DC. RESULTS: Our data shows that the mannose receptor, a C type lectin expressed by DC, acts as a major receptor for a number of allergens from diverse sources and that the level of allergen uptake is directly linked to the degree of the allergen glycosylation. We have also shown that down-regulation of the mannose receptor or the use of deglycosylated allergens leads to a significant reduction in allergen uptake (n 5 5, P < 0.05) by DC. CONCLUSIONS: The mannose receptor appears to be involved in the uptake of different allergens by human DC, and that the glycosylation state of the allergen is likely to play an important role in this process.
MONDAY
803