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Principal Investigator: Geoffrey Mueller
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2020
Award: $666,036
Funding agency: National Institute of Environmental Health Sciences
I. Origins of Allergic Disease
Over 100 million people worldwide suffer from birch pollen allergy. However, identification of molecular determinants driving the allergic responses to Bet v 1, the major birch pollen allergen, remains elusive. In collaboration with a group in Salzburg, Austria, we examined the pollen microbiota and investigated the allergenicity of Bet v 1 upon interaction with pollen-derived compounds. Sensitization to Bet v 1 is induced by an as-yet-undetermined pollen compound or mechanism in the pollen environment. These data suggest that sensitization is not exclusively linked to the intrinsic properties of individual proteins. In our future studies we will continue to investigate the birch pollen metabalome for evidence of small molecules that skew the immune response to allergy. Researchers in Salzburg will identify pollen fractions with intrinsic allergencity and provide them to NIEHS for analysis.
The metabolome of pollens is also potentially useful in identifying pollen taxa. The daily pollen forecast provides crucial information for allergic patients to avoid exposure to specific pollen. Pollen counts are typically measured with air samplers and analyzed with microscopy by trained experts. In contrast, we evaluated the effectiveness of identifying the component pollens using the metabolites extracted from an air-sampled pollen mixture. Our study demonstrated that NMR spectra of air-sampled pollen extracts can be used in an automated fashion to provide taxa and type-specific measures of the daily pollen count using deep learning algorithms.
II. Characterization of Allergens
A common general question about allergens is whether or not there are common properties. Several anecdotal studies have suggested that abundance in the allergic source, and protein stability are common among allergens. In our study we utilized 2 omics-based methods to address this question with statistical rigor. First we studied RNA-seq data from cockroaches, birch pollen, grass pollen, and ragweed pollen to conclude that allergens are among the most highly expressed proteins. Second, we used a mass spectrometry based approach to measure the stability of many proteins from these sources. When we combined the data with previous studies on the dust mite proteome, we could concluded that allergens were among the most stable and highly expressed from their natural sources. The important implications of this are in the design of therapies. The data suggests that destabilized allergens as therapeutics would be less likely to generate the dangerous allergic reactions, and hopefully promote tolerant responses.
Western blots and mass spectrometry were used to show the presence of the LS in peanut and walnut seeds. The NMR Structure of the Ara h 1 LS was determined and the IgE binding sites were modeled on this structure.
The epitopes with the highest degree of IgE binding were clustered within regions that were near cysteine residues. Of the patients tested, 96% showed IgE binding to those epitopes even if they recognized no other epitopes in the Ara h 1 LS. The NMR structure showed 4 of the cysteine residues are disulfide bonded and hold together two parallel alpha helices. IgE binding is shown to be located at the junction of the c-terminal region of the alpha helices and the beginning of each flexible loop. The results indicate that cysteine residues known to confer high structural stability to allergens may also coincide with areas of increased IgE binding frequency and intensity in Ara h 1 LS. The leader sequence contain multiple immunodominant epitopes and may be important in cross-reactivity and nut allergy.
At NIEHS we continue to characterize more leader sequence allergens from walnut, peanut, and cashew to better understand patient cross-reactivity.
The cockroach allergen Bla g 1 is known to bind hydrophobic ligands, but why the protein is an important allergen is unknown. We investigated a homolog of Bla g 1 from mosquitos called AZ1. This protein is upregulated in mosquitos following a blood feed, and is upregulated in response to viral infections. We have discovered that AZ1 can facilitate red blood cell lysis, and blocks infections of flaviviruses. The common mechanism of action is a lipid exchange, whereby AZ1 loaded with fatty acids will strip a PC molecule from either a red blood cell, or an enveloped virus and deliver the fatty acid, destabilizing the target membrane. This has important implications as a possible broad spectrum anti-viral agent. In returning to the question of Bla g 1 related allergy, lung surfactant also contains a high concentration of PC. Therefore we propose in the future to investigate if the lipid exchange properties of Bla g 1, facilitate lung damage that encourages allergic disease, or asthma exacerbations.
III. Adaptive Immune response
The defining characteristic of allergy is the generation of IgE antibodies, which leads to patient symptoms. We wish to probe more fundamental properties of the antibody response. It is suggested that by better understanding and characterizing the antibodies of all types new treatment modalities, or safer therapies can be developed.
Previously, we determined the first structure of the major peanut allergen Ara h 2. Ara h 2 is recognized by more than 90% of peanut allergic patients and sensitivity to Ara h 2 is measurable risk factor for peanut induced anaphylaxis. We have initiated a collaboration with researchers at Harvard University who have been studying the antibody production of peanut allergic patients in response to oral immunotherapy. By isolating B-cells from the patients and sequencing immunoglobulin genes, they found evidence that separate patients were honing in on similar regions of Ara h 2. We have received 5 of these antibody clones and are working to determine structures of antibody fragments in complex with Ara h 2. So far, we tested the production of all 5, and have scaled up production. The epitope information derived from these complexes will be useful in understanding the targets of the adaptive immune response of patients during peanut immunotherapy.
Directly examining human IgE in complex an allergen is technically challenging for a number of reasons. First of all, the memory cells that make IgE are extremely rare in sera. Scott Smith at Vanderbilt University has recently developed a technique to clone these rare cells and produce full length human IgE. We have acquired 4 human monoclonal IgE antibodies against the major dust mite allergen Der p 2 to study as a collaborative project. In the past year we have been measuring the interactions of the human IgE with Der p 2 via NMR technologies. We hope that this pioneering technique could be generalized to study the response to other allergens, and may be applicable to other antibody types besides IgE. The results will help us better understand the human immune response to allergens like Der p 2 with a hope using the information to design hypo-allergens that will improve allergy immunotherapy, better known as allergy-shots.
This project involves research on human coronavirus, novel coronavirus, COVID-19, Severe Acute Respiratory Syndrome coronavirus disease, SARS coronavirus, SARS-coronavirus-2, SARS-cov-2, SARS-cov2, SARS-related coronavirus 2, Severe acute respiratory syndrome coronavirus 2, SARS-Associated Coronavirus, SARS-cov, or SARS-Related Coronavirus.
Terms: <2019 novel coronavirus><2019-nCoV><Ab response><Address><Allergen Immunotherapy><Allergens><Allergic><Allergic Disease><Allergic Reaction><Allergy><Allergy Shot><Allergy immunotherapy><Ambrosia><Anaphylactic Reaction><Anaphylactic Shock><Anaphylaxis><Antibodies><Antibody Formation><Antibody Fragments><Antibody Production><Antibody Response><Antigenic Determinants><Antiviral Agents><Antiviral Drugs><Antivirals><Area><Austria><Automobile Driving><B blood cells><B cell><B cells><B-Cells><B-Lymphocytes><B-cell><Betula><Betula Genus><Binding><Binding Determinants><Binding Sites><Birch><Bla g 1><Blood><Blood Reticuloendothelial System><Blood erythrocyte><C-terminal><COVID-19><COVID19><Cashew><Cashew nut><Cell Body><Cells><Characteristics><CoV emergence><Cockroaches><Collaborations><Combining Site><Complex><Culicidae><Cysteine><Cytolysis><Dangerousness><Data><Der p 2><Der p 2 allergen><Der p II><Der p II allergen><Dermatophagoides Allergens><Dermatophagoides pteronyssinus antigen p 2><Diagnosis><Dictyoptera><Environment><Epitopes><Erythrocytes><Erythrocytic><Exposure to><Fatty Acids><Flavivirus Infections><Frequencies><Future><GeneHomolog><Generations><Goals><HCoV><Half-Cystine><Health><Homolog><Homologous Gene><Homologue><House Dust Mite Allergens><Hu-mABs><Human><Hydrophobicity><Hypersensitivity><Ig Genes><IgE><Immune mediated therapy><Immune response><Immunodominant Antigenic Determinants><Immunodominant Determinants><Immunodominant Domains><Immunodominant Epitopes><Immunodominant Regions><Immunodominant Sites><Immunoglobulin E><Immunoglobulin Fragments><Immunoglobulin Genes><Immunological response><Immunologically Directed Therapy><Immunotherapy><Individual><Investigators><Juglans><L-Cysteine><Length><Ligands><Link><Lipids><Lung Surfactant><Lung damage><Lysis><Marrow erythrocyte><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum 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coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe acute respiratory syndrome coronavirus 2><Source><Structure><Symptoms><Techniques><Technology><Testing><Therapeutic><Training><Tree Nut Allergy><Tree Nut Hypersensitivity><Universities><Viral Diseases><Virus Diseases><Walnut><Western Blotting><Western Immunoblotting><Wuhan coronavirus><adaptive immune response><air sampling><allergen Bla g 1><allergen response><allergic response><allergic to nuts><allergy response><allergy to nuts><alpha helix><anti-viral agents><anti-viral drugs><anti-virals><antibody biosynthesis><asthma attack><asthma exacerbation><base><blood corpuscles><cockroach allergen><corona virus disease 2019><coronavirus disease 2019><coronavirus emergence><cross reactivity><deep learning algorithm><design><designing><disulfide bond><driving><dust mite><dust mite allergens><effectiveness evaluation><emergent CoV><emergent coronavirus><emerging CoV><emerging coronavirus><evaluate 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up><seed><severe acute respiratory syndrome-CoV><small molecule><therapy design><transcriptome sequencing><treatment design><viral infection><virus envelope><virus infection><virus-induced disease><α-helix>