Molecular Allergy

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

I. Origins of Allergic Disease The cockroach allergen Bla g 1 encloses an exceptionally large hydrophobic cavity, which allows it to bind and deliver unsaturated fatty acid ligands. Bla g 1-mediated delivery of naturally occurring (nMix) ligands has been shown to destabilize lipid membranes, contributing to its digestive/antiviral functions within the source organism. However, the consequences of this activity on Bla g 1 allergenicity following human exposure remain unknown. In this work, we show that Bla g 1-mediated membrane disruption can induce a proinflammatory immune response in mammalian cells via two complementary pathways. At high concentrations, the cytotoxic activity of Bla g 1 induces the release of proinflammatory cytosolic contents including damage-associated molecular patterns (DAMPs) such as heat-shock Protein-70 (HSP70) and the cytokine interleukin-1 (IL-1β). Sublytic concentrations of Bla g 1 enhanced the ability of phospholipase A2 (PLA2) to extract and hydrolyze phospholipid substrates from cellular membranes, stimulating the production of free polyunsaturated fatty acids (PUFAs) and various downstream inflammatory lipid mediators. Both of these effects are dependent on the presence of Bla g 1's natural fatty-acid (nMix) ligands with CC50 values corresponding to the concentrations required for membrane destabilization reported in previous studies. Taken together, these results suggest that mechanisms through which Bla g 1-mediated lipid delivery and membrane destabilization could directly contribute to cockroach allergic sensitization. Current work in the lab is focused on whether or the membrane disruption properties can be extended to other lipid binding allergens. This could explain why certain sources and allergens are potently immunogenic. II. Characterization of Allergens In peanut allergy, Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea 6 (Ara h 6) are two clinically relevant peanut allergens with known structural and sequence homology and demonstrated cross-reactivity. We have previously utilized X-ray crystallography and epitope binning to define the epitopes on Ara h 2. We aimed to quantitatively characterize the cross-reactivity between Ara h 2 and Ara h 6 on a molecular level using human monoclonal antibodies (mAbs) and structural characterization of allergenic epitopes. We utilized mAbs cloned from Ara h 2 positive single B cells isolated from peanut-allergic, oral immunotherapy-treated patients to quantitatively analyze cross-reactivity between recombinant Ara h 2 (rAra h 2) and Ara h 6 (rAra h 6). Overall, mAb affinity was significantly lower to rAra h 6 than it was to rAra h 2. This difference in affinity was primarily due to increased dissociation of the antibodies from rAra h 6, a phenomenon explained by the higher conformational flexibility of the Ara h 6-antibody complexes in comparison to Ara h 2-antibody complexes. Our results further elucidate the cross-reactivity of peanut 2S albumins on a molecular level and support the clinical immunodominance of Ara h 2. Current work has been focused on understanding the properties of germ-line antibodies where the mature antibody is cross-reactive but the germ-line antibody is not. This data will provide information on how antibodies mature in response to repeated exposure to the peanut allergens. Peanut and tree nut allergies are frequently comorbid, even though peanuts and tree nuts are actually distantly related. Under current theory, this would not predict the extensive comorbidity observed. We suggest that the alpha-hairpinin domains, usually associated with vicilin allergens are likely a cause of the observed cross-reactive antibodies. Work in the lab this year has better defined the differentiation between antibodies to the vicilin and alpha-hairpinin domain from the peanut allergen Ara h 1. Further we’ve characterized the cross-reactivity of peanut, tree nut, and nightshade derived alpha-hairpinin domains, which will hopefully help explain cross-reactivity patterns observed in patients. Last, we have discovered two atypical cross-linking situations, whereby a single antibody can activate anaphylaxis in a mouse model using Ara h 1 and Jug r 2. These cases may illustrate why peanut and tree nut allergens are frequently potent activators of anaphylactic reactions. NsLTP allergens are common plant allergens that appear to have extensive cross-reactivity across many plant species. However, with patient serum it is difficult to untangle if there are many specific antibodies, or a few highly promiscuous antibodies. We have discovered a human IgG4 antibody that is highly cross-reactive to 19 different nsLTPs and structurally characterized the binding. The result explained how an antibody could be so cross-reactive. This anecdotally supports the notion that a few antibodies could be highly promiscuous. III. Adaptive Immune response Antibodies can evolve to recognize a diverse array of foreign proteins. In our investigations of antibodies to the peanut allergen Ara h 2, we noticed that most of the antibodies were directed against a specific epitope. In work published this year, we traced back the origins of these antibodies to see how they evolved. Based on our structural characterization we developed mutant proteins to probe how many people had these specific antibodies. We surprisingly found that high-affinity antibodies to patients are actually germ-line encoded, and they are surprisingly common in the population. The implications for disease are that it seems very easy to make anti-peanut antibodies. Future work will need to assess what is the trigger that causes these antibodies to recognize peanut proteins as potentially pathogenic. In previous years we have developed and patented hypoallergenic forms of allergens by making mutant peanut proteins. Hypoallergens are suggested to make allergy immunotherapy less dangerous, and more efficacious. This year we explored a novel method of making hypoallergens by coupling gold nanoparticles to egg allergens. This method has facile chemistry and could be generally applicable to other allergens.