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Bla g 2 Hypoallergens Retaining the Native Fold and Capacity to Modulate T Cell Reactivity Provide Candidates for Cockroach Immunotherapy
Abstracts AB165
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
Bla g 2 Hypoallergens Retaining the Native Fold and Capacity to Modulate T Cell Reactivity Provide Candidates for Cockroach Immunotherapy Anna Pomes, PhD, FAAAAI1, Jill Glesner, BS1, Judith A. Woodfolk, MBChB, PhD, FAAAAI2, Paul Wright, BS3, Christopher L. Kepley, PhD, MBA, FAAAI4, Mi Li, MS5, Martin Himly, PhD6, Alla Gustchina, PhD5, Alexander Wlodawer, PhD5, Martin D. Chapman, PhD, FAAAAI1; 1Indoor Biotechnologies, Inc., Charlottesville, VA, 2Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 3University of Virginia, Charlottesville, VA, 4 Joint School of Nanoscience and Nanoengineering, University of North Carolina, Greensboro, NC, 5National Cancer Institute, Frederick, MD, 6 University of Salzburg, Salzburg, Austria. RATIONALE: Modified allergens that display reduced IgE reactivity along with T-cell activating properties, are strong candidates for immunotherapy, because of the potential to decrease side-effects due to IgE crosslinking, but still retain immunogenicity. METHODS: Single and multiple Bla g 2 mutants were designed according to prior knowledge of the antigenic structure of the allergen and expressed in Pichia pastoris. Folding of the mutants was assessed by CD spectrometry or X-ray crystallography. IgE reactivity was measured by antibody binding and mast cell release assays. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and CD4+ T-cell phenotype in PBMC cultures. RESULTS: Single and multiple mutations of residues implicated in binding to monoclonal antibodies (K132A, K251A and/or F162Y) reduced IgE reactivity but did not influence the native molecular fold, as proven by comparing the triple mutant with wild type Bla g 2 by X-ray crystallography. As compared with wild type allergen, mutants KK and KKF showed from at least 100-fold to a total reduction in IgE antibody binding. Whereas similar T-cell activating capacity was retained based on CD25 expression, both mutants were weaker inducers of the Th2 cytokine, IL-13. Furthermore, both mutants induced high levels of IL-10 from a non-Tcell source, and levels induced by the triple mutant exceeded those induced by Bla g 2 (p50.004). CONCLUSIONS: A rational design of site-directed mutagenesis was effective in producing candidate molecules for immunotherapy that maintain the same fold as wild type Bla g 2, but display reduced IgE reactivity with T-cell modulatory potential.
537
Protective Role of Hydrogen Sulfide in Paramyxovirus Infection John P. Kelley, MD, Hui Li, PhD, Yinghong Ma, Teodora Ivanciuc, PhD, Narayana Komaravelli, PhD, Ciro Coletta, PhD, Csaba Szabo, PhD, Roberto P. Garofalo, MD, Antonella Casola, M.D.; University of Texas Medical Branch, Galveston, TX. RATIONALE: Hydrogen sulfide (H2S) is a novel gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases, but its role in viral-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are major causes of upper and lower respiratory tract infections in children, for which no vaccine or effective treatment is available. We hypothesized that administration of H2S during RSVand hMPV infection would reduce viral-induced proinflammatory mediator release and viral replication. METHODS: Airway epithelial cells were infected with RSV or hMPV, treated with either the slow-releasing H2S donor GYY4137 or propargylglysin (PAG), an inhibitor of intracellular H2S production, and harvested to measure cytokine and chemokine secretion, and viral titers. In vivo efficacy was tested in a mouse model of RSV infection treated with GYY4137 by assessing viral replication and disease. RESULTS: RSV- and hMPV-induced secretion of several cytokines and chemokines, such as IL-8 and RANTES, and viral titers were significantly decreased by GYY4137 treatment, and increased by PAG. Infected mice treated with GYY4137 showed reduced viral titers and attenuated RSV-
induced body weight loss, with faster recovery, compared to the untreated mice. CONCLUSIONS: Modulation of cellular H2S significantly impacts cellular responses and viral replication in an in vitro and in vivo model of RSV/hMPV infection. Our results underscore an important role of H2S in regulating virus infection and host defenses that could lead to a novel treatment strategy for paramyxovirus infections, and possibly other respiratory viral infections.
538
Accurate Assessment of Personal Air Pollutant Exposures in Inner-City Asthmatic Children Cullen M. Dutmer, MD1,2, Allison M. Schiltz, BA1, Anna Faino, MS1, Nathan Rabinovitch, MD, MPH1, Seung-Hyun Cho, PhD3, Ryan T. Chartier, MS3, Charles E. Rodes, PhD3, Jonathan W. Thornburg, PhD3, Andrew H. Liu, MD, FAAAAI1,2; 1National Jewish Health, Denver, CO, 2 University of Colorado School of Medicine, Aurora, CO, 3RTI International, Research Triangle Park, NC. RATIONALE: Inner-city environments seem particularly toxic for children with asthma, in part due to air pollutant exposures. Personal monitors that sample from an individual’s ‘‘breathing zone’’ offer a more accurate method than stationary monitors to measure air pollutant exposures and minimize misclassification bias. METHODS: We performed an observational study to measure inner-city children’s exposure to PM10 (particulate matter<10mm), black carbon (BC), and brown carbon (BrC) using the MicroPEMä (RTI International), and to NO2 using an Ogawaä passive badge (Ogawa USA). Fifteen inner-city children (8-15 years) participated in this study, including 12 asthmatics. Eight participants reported exposure to cigarette smokers. Participants were instructed to wear personal monitors during waking hours. Stationary monitors (PEMä, MSP Corp.) were installed into each participant’s bedroom. Targeted sampling period was 72 hours. Exposure levels between personal and stationary monitors were compared via linear mixed models with random intercepts for sibling pairs. RESULTS: Waking wearing compliance (WWC) was excellent in 87% of participants, with a median WWC of 83% for those with acceptable WWC (>60%). Personal monitor levels were significantly higher than, and correlated variably with, stationary monitor levels for PM10 (>30% higher, p50.023; intraclass correlation coefficient (ICC)50.342, 95% confidence interval [-0.254;0.399]), BC (>7-fold higher, p<0.0001; ICC50.082 [-0.429;0.594]), and BrC (>4-fold higher, p<0.0001; ICC50.635 [0.166;1.103]). NO2 levels did not differ significantly. CONCLUSIONS: Accurate exposure assessment using personal exposure monitors, such as the MicroPEMä, are feasible for use with inner-city children. Stationary monitors inaccurately estimate personal exposure to PM, as they do not capture the higher concentrations found near strong PM emission sources.
SUNDAY
536
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
Bla g 2 Hypoallergens Retaining the Native Fold and Capacity to Modulate T Cell Reactivity Provide Candidates for Cockroach Immunotherapy Anna Pomes, PhD, FAAAAI1, Jill Glesner, BS1, Judith A. Woodfolk, MBChB, PhD, FAAAAI2, Paul Wright, BS3, Christopher L. Kepley, PhD, MBA, FAAAI4, Mi Li, MS5, Martin Himly, PhD6, Alla Gustchina, PhD5, Alexander Wlodawer, PhD5, Martin D. Chapman, PhD, FAAAAI1; 1Indoor Biotechnologies, Inc., Charlottesville, VA, 2Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 3University of Virginia, Charlottesville, VA, 4 Joint School of Nanoscience and Nanoengineering, University of North Carolina, Greensboro, NC, 5National Cancer Institute, Frederick, MD, 6 University of Salzburg, Salzburg, Austria. RATIONALE: Modified allergens that display reduced IgE reactivity along with T-cell activating properties, are strong candidates for immunotherapy, because of the potential to decrease side-effects due to IgE crosslinking, but still retain immunogenicity. METHODS: Single and multiple Bla g 2 mutants were designed according to prior knowledge of the antigenic structure of the allergen and expressed in Pichia pastoris. Folding of the mutants was assessed by CD spectrometry or X-ray crystallography. IgE reactivity was measured by antibody binding and mast cell release assays. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and CD4+ T-cell phenotype in PBMC cultures. RESULTS: Single and multiple mutations of residues implicated in binding to monoclonal antibodies (K132A, K251A and/or F162Y) reduced IgE reactivity but did not influence the native molecular fold, as proven by comparing the triple mutant with wild type Bla g 2 by X-ray crystallography. As compared with wild type allergen, mutants KK and KKF showed from at least 100-fold to a total reduction in IgE antibody binding. Whereas similar T-cell activating capacity was retained based on CD25 expression, both mutants were weaker inducers of the Th2 cytokine, IL-13. Furthermore, both mutants induced high levels of IL-10 from a non-Tcell source, and levels induced by the triple mutant exceeded those induced by Bla g 2 (p50.004). CONCLUSIONS: A rational design of site-directed mutagenesis was effective in producing candidate molecules for immunotherapy that maintain the same fold as wild type Bla g 2, but display reduced IgE reactivity with T-cell modulatory potential.
537
Protective Role of Hydrogen Sulfide in Paramyxovirus Infection John P. Kelley, MD, Hui Li, PhD, Yinghong Ma, Teodora Ivanciuc, PhD, Narayana Komaravelli, PhD, Ciro Coletta, PhD, Csaba Szabo, PhD, Roberto P. Garofalo, MD, Antonella Casola, M.D.; University of Texas Medical Branch, Galveston, TX. RATIONALE: Hydrogen sulfide (H2S) is a novel gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases, but its role in viral-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are major causes of upper and lower respiratory tract infections in children, for which no vaccine or effective treatment is available. We hypothesized that administration of H2S during RSVand hMPV infection would reduce viral-induced proinflammatory mediator release and viral replication. METHODS: Airway epithelial cells were infected with RSV or hMPV, treated with either the slow-releasing H2S donor GYY4137 or propargylglysin (PAG), an inhibitor of intracellular H2S production, and harvested to measure cytokine and chemokine secretion, and viral titers. In vivo efficacy was tested in a mouse model of RSV infection treated with GYY4137 by assessing viral replication and disease. RESULTS: RSV- and hMPV-induced secretion of several cytokines and chemokines, such as IL-8 and RANTES, and viral titers were significantly decreased by GYY4137 treatment, and increased by PAG. Infected mice treated with GYY4137 showed reduced viral titers and attenuated RSV-
induced body weight loss, with faster recovery, compared to the untreated mice. CONCLUSIONS: Modulation of cellular H2S significantly impacts cellular responses and viral replication in an in vitro and in vivo model of RSV/hMPV infection. Our results underscore an important role of H2S in regulating virus infection and host defenses that could lead to a novel treatment strategy for paramyxovirus infections, and possibly other respiratory viral infections.
538
Accurate Assessment of Personal Air Pollutant Exposures in Inner-City Asthmatic Children Cullen M. Dutmer, MD1,2, Allison M. Schiltz, BA1, Anna Faino, MS1, Nathan Rabinovitch, MD, MPH1, Seung-Hyun Cho, PhD3, Ryan T. Chartier, MS3, Charles E. Rodes, PhD3, Jonathan W. Thornburg, PhD3, Andrew H. Liu, MD, FAAAAI1,2; 1National Jewish Health, Denver, CO, 2 University of Colorado School of Medicine, Aurora, CO, 3RTI International, Research Triangle Park, NC. RATIONALE: Inner-city environments seem particularly toxic for children with asthma, in part due to air pollutant exposures. Personal monitors that sample from an individual’s ‘‘breathing zone’’ offer a more accurate method than stationary monitors to measure air pollutant exposures and minimize misclassification bias. METHODS: We performed an observational study to measure inner-city children’s exposure to PM10 (particulate matter<10mm), black carbon (BC), and brown carbon (BrC) using the MicroPEMä (RTI International), and to NO2 using an Ogawaä passive badge (Ogawa USA). Fifteen inner-city children (8-15 years) participated in this study, including 12 asthmatics. Eight participants reported exposure to cigarette smokers. Participants were instructed to wear personal monitors during waking hours. Stationary monitors (PEMä, MSP Corp.) were installed into each participant’s bedroom. Targeted sampling period was 72 hours. Exposure levels between personal and stationary monitors were compared via linear mixed models with random intercepts for sibling pairs. RESULTS: Waking wearing compliance (WWC) was excellent in 87% of participants, with a median WWC of 83% for those with acceptable WWC (>60%). Personal monitor levels were significantly higher than, and correlated variably with, stationary monitor levels for PM10 (>30% higher, p50.023; intraclass correlation coefficient (ICC)50.342, 95% confidence interval [-0.254;0.399]), BC (>7-fold higher, p<0.0001; ICC50.082 [-0.429;0.594]), and BrC (>4-fold higher, p<0.0001; ICC50.635 [0.166;1.103]). NO2 levels did not differ significantly. CONCLUSIONS: Accurate exposure assessment using personal exposure monitors, such as the MicroPEMä, are feasible for use with inner-city children. Stationary monitors inaccurately estimate personal exposure to PM, as they do not capture the higher concentrations found near strong PM emission sources.
SUNDAY
536