Molecular Allergy

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

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. Using recently-developed high-throughput approaches, both denaturant-dependent thermolysin digestion, and time-dependent limited proteolysis with Cathepsin S (the endosomal protease) were used to the measure the stabilities of proteins extracted from birch pollen. Again, the allergens were found to be simultaneously more abundant and more resistant to proteolysis. We found that Bet v 1, with the highest prevalence in birch allergic patients, is among the most abundant and proteolytically resistant proteins. Thus, stability and abundance appear highly correlated with allergic sensitization in accord with a plausible biological mechanism. In the future it may be interesting to repeat these studies using environmental samples of household dust with and without pets since animal dander is a class of allergens we have not examined. Or it may be useful to compare urban pollen samples to rural pollen samples to see if reports on the nitrosylation of Bet v 1 due to pollution affect the stability of Bet v 1 or other allergens. Undoubtedly, there are additional factors besides stability and abundance that influence sensitization that will motivate future studies. II. Characterization of Allergens The NMR Structure of the Ara h 1 LS and Jug r 2 LS 1, 2, and 3 were 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, pistachio and cashew to better understand patient cross-reactivity. A common general question about allergens is whether or not there are common properties. 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 and corona viruses related to the COVID-19 virus. 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. During this review period we examined the ability of AZ1 to deliver non-natural anti-viral ligands to prevent infection of SAR-COV-2. 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 human monoclonal IgE antibodies against the LS allergens mentioned above. In the past year we have been measuring the interactions of the human IgE with Can f 1 and Fel d 7 via NMR technologies. 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. To better understand the antibody respond, we studied in detail a nanobody to green fluorescent protein using NMR and molecular modeling techniques. These studies benchmarked new computational techniques with physical NMR data. Data from this model system will be applicable to future modeling studies of antibody evolution.