Sara Huber

Amb a 1 isoforms: unequal siblings with distinct immunological features

Ragweed pollen represents a major allergy risk factor. Ragweed extracts contain five different isoforms of the major allergen Amb a 1. However, the immunological characteristics of Amb a 1 isoforms are not fully investigated. Here, we compared the physicochemical and immunological properties of three most important Amb a 1 isoforms.

The Evolution of Human Basophil Biology from Neglect towards Understanding of Their Immune Functions

Being discovered long ago basophils have been neglected for more than a century. During the past decade evidence emerged that basophils share features of innate and adaptive immunity. Nowadays, basophils are best known for their striking effector role in the allergic reaction. They hence have been used for establishing new diagnostic tests and therapeutic approaches and for characterizing natural and recombinant allergens as well as hypoallergens, which display lower or diminished IgE-binding activity. However, it was a long way from discovery in 1879 until identification of their function in hypersensitivity reactions, including adverse drug reactions. Starting with a historical background, this review highlights the modern view on basophil biology.

Context matters: TH2 polarization resulting from pollen composition and not from protein-intrinsic allergenicity

In contrast to the pollen context, the major allergen Bet v 1 alone is not responsible for Th2 polarization in allergic sensitization. These findings are highly relevant for birch pollen allergy prophylaxis.

Expression and Characterization of Functional Recombinant Bet v 1.0101 in the Chloroplast of Chlamydomonas reinhardtii

Background: Allergen immunotherapy (AIT) still plays a minor role in the treatment of allergic diseases. To improve the acceptance of AIT by allergic patients, the treatment has to become more convenient and efficacious. One possibility is the oral application of allergens or derivatives thereof. Therefore, we sought to produce a recombinant allergen in the green alga Chlamydomonas reinhardtii as a novel production platform. Methods: The major birch pollen allergen Bet v 1 was selected as candidate molecule, and a codon-optimized gene was synthesized and stably integrated into the microalga C. reinhardtii FUD50. Positive transformants were identified by PCR, cultured, and thereafter cells were disrupted by sonication. Bet v 1 was purified from algal total soluble protein (TSP) by affinity chromatography and characterized physicochemically as well as immunologically. Results: All transformants showed expression of the allergen with yields between 0.01 and 0.04% of TSP. Algal-derived Bet v 1 displayed similar secondary structure elements as the Escherichia coli-produced reference allergen. Moreover, Bet v 1 produced in C. reinhardtii showed binding comparable to human IgE as well as murine Bet v 1-specific IgG. Conclusion: We could successfully produce recombinant Bet v 1 in C. reinhardtii. As microalgae are classified as GRAS (generally recognized as safe), the pilot study supports the development of novel allergy treatment concepts such as the oral administration of allergen-containing algal extracts for therapy.

Does clinical outcome of birch pollen immunotherapy relate to induction of blocking antibodies preventing IgE from allergen binding? A pilot study monitoring responses during first year of AIT

BackgroundThe clinical benefit of allergen-specific immunotherapy (AIT) involves induction of blocking antibodies. It is not clear if these antibodies function via steric hindrance alone or a combination of levels, avidities, and epitope specificities, and clinical outcome cannot be predicted. We aim to in-depth characterize serum antibody profiles during birch pollen AIT, investigate therapy-induced antibodies for their capacity to block IgE binding to Bet v 1 and correlate data with clinical outcomes.MethodsImmune responses of five birch pollen allergic patients were monitored during the first year of AIT by nasal provocation tests (NPTs), ImmunoCAP, immunoblots, direct and avidity enzyme-linked immunosorbent assays, mediator release assays, facilitated antigen binding (FAB) assays, and inhibition mediator release assays.ResultsThere was no correlation between NPT results and therapy-induced changes in levels (IgE, IgG, IgA, IgM), avidities, or mediator release potency of Bet v 1-specific antibodies. In FAB assays, blocking antibodies initiated upon AIT were shown to prevent formation of Bet v 1-IgE complexes of an indicator serum pool and significantly correlated with clinical readout. Inhibition mediator release assays using patient-specific IgE for passive sensitization revealed therapy-induced blocking capacities with very good correlation to NPT results. Notably, this assay was the only one to detect a non-responder during treatment in this pilot study.ConclusionsClinical outcome of AIT depends on induction of blocking antibodies able to prevent the patient’s own IgE from allergen binding. Monitoring of clinical efficacy seems to be best achieved using the inhibition mediator release assay, as development of relevant blocking antibodies can be verified in a patient-tailored manner.

Two Distinct Conformations in Bet v 2 Determine Its Proteolytic Resistance to Cathepsin S

Birch pollen allergy affects more than 20% of the European allergic population. On a molecular level, birch pollen allergy can be linked to the two dominant allergens Bet v 1 and Bet v 2. Bet v 2 belongs to the profilin family, which is abundant in the plant kingdom. Importantly, the homologous plant profilins have a conserved cysteine motif with a currently unknown functional relevance. In particular, it is unknown whether the motif is relevant for disulfide formation and to what extent it would affect the profilins’ structural, functional and immunological properties. Here we present crystal structures of Bet v 2 in the reduced and the oxidized state, i.e., without and with a disulfide bridge. Despite overall structural similarity, the two structures distinctly differ at their termini which are stabilized to each other in the oxidized, i.e., disulfide-linked state. These structural differences translate into differences in their proteolytic resistance. Whereas the oxidized Bet v 2 is rather resistant towards the endolysosomal protease cathepsin S, it is rapidly degraded in the reduced form. By contrast, both Bet v 2 forms exhibit similar immunological properties as evidenced by their binding to IgE antibodies from birch pollen allergic patients and by their ability to trigger histamine release in a humanized rat basophilic leukemia cells (RBL) assay, independent of the presence or absence of the disulfide bridge. Taken together our findings suggest that the oxidized Bet v 2 conformation should be the relevant species, with a much longer retention time to trigger immune responses.