Mattias Levin

The human IgE repertoire

IgE is a key mediator in allergic diseases. However, in strong contrast to other antibody isotypes, many details of the composition of the human IgE repertoire are poorly defined. The low levels of human IgE in the circulation and the rarity of IgE-producing B cells are important reasons for this lack of knowledge. In this review, we summarize the current knowledge on these repertoires both in terms of their complexity and activity, i.e. knowledge which despite the difficulties encountered when studying the molecular details of human IgE has been acquired in recent years. We also take a look at likely future developments, for instance through improvements in sequencing technology and methodology that allow the isolation of additional allergen-specific human antibodies mimicking IgE, as this certainly will support our understanding of human IgE in the context of human disease in the years to come.

Human IgE against the major allergen Bet v 1 – defining an epitope with limited cross‐reactivity between different PR‐10 family proteins

The interaction between IgE and allergen is a key event at the initiation of an allergic response, and its characteristics have substantial effects on the clinical manifestation. Despite this, the molecular details of the interaction between human IgE and the major birch allergen Bet v 1, one of the most potent tree allergens, still remain poorly investigated.

Phl p 1–Specific Human Monoclonal IgE and Design of a Hypoallergenic Group 1 Grass Pollen Allergen Fragment

Detailed understanding of how Abs of the IgE isotype interact with allergen at the onset of an allergic reaction is of great importance for deciphering mechanisms involved in the development of disease and may aid in the design of hypoallergenic variants. In this study, we have used a set of human monoclonal IgE Abs derived from the repertoires of allergic individuals, specific for the major timothy grass pollen allergen Phl p 1, to gain detailed information on the interaction between Abs and allergen. These allergen-specific IgE are to varying degrees cross-reactive toward both different allergen isoforms and various group 1 allergens originating from other grass species. The usage of human monoclonal IgE, as an alternative to polyclonal preparations or mouse Abs, allowed us to locate several important IgE-binding epitopes on the C-terminal domain of Phl p 1, all clustered to an IgE-binding “hot spot.” By introducing three mutations in the IgE-binding area of the C-terminal domain we were able to significantly reduce its reactivity with serum IgE. In conclusion, our study shows the great potential of using human monoclonal IgE as a tool for studies of the molecular interactions taking place during allergic responses. Furthermore, we present a novel IgE-hyporeactive fragment with the potential to be used as a safer hypoallergenic alternative in specific immunotherapy than the pollen extracts used today.

Diversity of immunoglobulin E‐encoding transcripts in sinus mucosa of subjects diagnosed with non‐allergic fungal eosinophilic sinusitis

Cite this as: M. Levin, L. W. Tan, L. Baker, P.‐J. Wormald, L. Greiff and M. Ohlin, Clinical & Experimental Allergy, 2011 (41) 811–820.

Inconclusive Evidence for or against Positive Antigen Selection in the Shaping of Human Immunoglobulin E Repertoires: A Call for New Approaches.

Background: The mechanisms driving the development of immunoglobulin E (IgE) antibody repertoires are a matter of debate. Alternatives to the classical view on antibody development, involving somatic mutation and antigen-driven selection of high-affinity variants in germinal centers, have been proposed. Methods: We have re-analyzed the pattern of mutations in previously isolated and characterized human clonally unrelated IgE-encoding transcripts using the validated focused binomial methodology to find evidence in such genes of antigen-specific selection. Results: As expected there is a selection against replacement mutations in IgE framework regions. In contrast, in all examined cases but one (assessing IgE repertoires of parasite-infected individuals) there was no evidence in favor of either positive or negative selection in complementarity determining regions. Importantly, however, the validated method also failed to detect selection for replacement mutations in two, non-IgE, hypermutated antibody populations targeting tetanus toxoid and vaccinia virus, respectively. Conclusions: Current methodology is unable to define with certainty, using commonly assessed IgE repertoire sizes, whether antigen selection is or is not a major driving force in the establishment of human IgE. New approaches are needed to address this matter.

Multiple independent IgE epitopes on the highly allergenic grass pollen allergen Phl p 5.

Group 5 allergens are small proteins that consist of two domains. They belong to the most potent respiratory allergens.

A folded and immunogenic IgE-hyporeactive variant of the major allergen Phl p 1 produced in Escherichia coli.

BackgroundGroup 1 grass pollen allergens are a major cause of allergic disease. Specific immunotherapy involving controlled administration of allergens can be used as a disease-modifying treatment for such disease. Recombinant allergen variants with reduced IgE binding capacity may be used as component in such vaccines, as they may induce fewer treatment side effects than materials currently in use. A mutated variant of the immunodominant C-terminal domain of the group 1 grass pollen allergen Phl p 1 was recently established through an approach that used a set of human monoclonal IgE as a guide to identify mutations that disturbed IgE-allergen interactions. Further analysis of this domain is required to establish its potential for use in treatment.MethodsGST-tagged wild-type and mutated C-terminal domains of Phl p 1 were produced in Escherichia coli TUNER(DE3). The products were purified by affinity chromatography on immobilized glutathione. GST was removed by enzymatic cleavage and tag-free products were purified by size exclusion chromatography. Products were assessed by SDS-PAGE, circular dichroism spectroscopy, differential scanning fluorimetry and dynamic light scattering. Rats were immunized with GST-tagged and tag-free mutated C-terminal domain of Phl p 1. Antigen-binding properties of induced antibodies were assessed by immunochemical analysis.ResultsThe mutated domain has a structure very similar to that of the wild-type domain as determined by circular dichroism, but a reduced thermal stability. Immunization of rats demonstrates that this IgE-hyporeactive domain, despite its three sequence modifications (K8A, N11A, D55A), is able to induce antibodies that substantially block the binding of allergic subjects’ IgE to the wild-type allergen.ConclusionsIt is concluded that this IgE-hyporeactive molecule can be produced in folded form and that it is able to induce an antibody response that efficiently competes with IgE recognition of Phl p 1. These findings suggest that it, or a further evolved variant thereof, is a candidate for use as a component in specific immunotherapy against grass pollen allergy.

The ΙgΕ-epitome viewed through the eyes of human monoclonal ΙgΕ

Background Despite the key role of IgE as the allergen recognizer at the initiation of allergic responses our knowledge on the molecular interactions between human IgE and allergen is limited. To date, most studies aiming at identification of IgE-reactive epitopes have had to rely on polyclonal IgE-preparations, non-human antibodies or antibodies of isotypes other than IgE. However, due to their intrinsic ability to recognize highly relevant, in the context of allergy, epitopes on allergens, allergen-specific human monoclonal IgE, represented by recombinant human antibody fragments, offer an opportunity to decipher the IgE-epitome of major allergens with precision and at high resolution. Such information may guide us in development of novel therapeutic candidates and better diagnostic tools. Methods We have isolated a large pool of antibody variable domain fragments, specific for several major allergens, from the IgE repertoires of allergic donors. Three sets of such binders, targeting the major birch pollen allergen Bet v 1 (n = 4) and the major timothy grass pollen allergens Phl p 1 (n = 5) and Phl p 5 (n = 10), and their interactions with respective allergen have been studied by immunochemical methodologies. Results We have, using our own technology and through collaborations, been able to define multiple non-overlapping IgE-reactive epitopes on all three investigated allergens. Two novel epitopes on Bet v 1 were identified, which allowed for identification of single amino acid residues with crucial importance for the interaction between human IgE and allergens in the PR-10 protein family. Further, it was shown that the relatively small allergen Phl p 5 carries a complex composition of IgE-reactive epitopes, providing an explanation to its high allergenicity. The less complex epitope composition on Phl p 1, on the other hand, with an IgE-binding hot-spot located on the surface of its immunodominant C-terminal domain, allowed us to, in a rational manner, design a hypoallergenic group 1 gras sp ollen allergen fragment, with the potential to be used as a safer alternative to the today used extracts in specific immunotherapy. Conclusion