Structural basis for cross-reactivity and conformation fluctuation of the major beech pollen allergen Fag s 1.

Fag s 1 is a member of the Pathogen Related protein family 10 (PR-10) and can elicit cross-reaction with IgE antibodies produced against the birch pollen allergen Bet v 1. The Nuclear Magnetic Resonance (NMR) structure of Fag s 1 is presented along with its dynamic properties. It shares 66% identity with Bet v 1 and exhibits the expected three α-helices and seven ß-sheets arranged as a semi-beta barrel and exposing the residues mapped as the Bet v 1 IgE epitope. The structural dynamics of Fag s 1 were monitored on the fast and intermediate timescales, using relaxation rates. The complex dynamics of Fag s 1 are closely related to the internal cavity, and they modulate IgE and ligand binding.

Harmonization of the Genetic Code Effectively Enhances the Recombinant Production of the Major Birch Pollen Allergen Bet v 1.

BACKGROUND: Enhancing the quality and yield of protein production in heterologous expression systems is an important issue for developing new biopharmaceuticals. It has been shown that the dynamics of protein folding is influenced by codon frequencies. As codon usage frequencies are species specific, this can affect heterologous protein expression. In this respect, "codon harmonization," that is, the usage of synonymous codons with usage frequencies in the host resembling the usage frequencies in the native organism, is a promising strategy. As recombinant proteins are important tools in the area of allergy research, we investigated in this study the influence of codon harmonization on the production of the major birch pollen allergen Bet v 1.0101. METHODS: To accomplish this task, parallel production of several batches of rBet v 1, BWT, together with a harmonized variant, BH, was applied. The expression yield of soluble and insoluble protein was assayed via densitometric analysis of -SDS-PAGEs for every batch. The quality of purified proteins was assessed with a variety of physicochemical methods including mass spectrometry, circular dichroism, dynamic light scattering, Fourier transform infrared spectroscopy, in vitro degradation, and 1-anilino-8-naphthalene sulfonate-binding assays. Patients' IgE reactivity was tested in enzyme-linked immunosorbent assays and rat basophil mediator release experiments. RESULTS: No significant differences in the ligand-binding capacity and secondary structure elements, as well as, in immunological assays could be found; however, the production yield was drastically increased for BH. CONCLUSION: We could show that codon harmonization is a powerful method to enhance protein yields in heterologous expression systems and should be considered especially for difficult-to-express proteins.

T Cell Epitope-Containing Domains of Ragweed Amb a 1 and Mugwort Art v 6 Modulate Immunologic Responses in Humans and Mice.

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy. METHODS: Domains of Amb a 1 (Amb a 1α) and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT. RESULTS: The IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected. CONCLUSION: Accumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.

Pollen Allergens for Molecular Diagnosis.

Pollen allergens are one of the main causes of type I allergies affecting up to 30% of the population in industrialized countries. Climatic changes affect the duration and intensity of pollen seasons and may together with pollution contribute to increased incidences of respiratory allergy and asthma. Allergenic grasses, trees, and weeds often present similar habitats and flowering periods compromising clinical anamnesis. Molecule-based approaches enable distinction between genuine sensitization and clinically mostly irrelevant IgE cross-reactivity due to, e. g., panallergens or carbohydrate determinants. In addition, sensitivity as well as specificity can be improved and lead to identification of the primary sensitizing source which is particularly beneficial regarding polysensitized patients. This review gives an overview on relevant pollen allergens and their usefulness in daily practice. Appropriate allergy diagnosis is directly influencing decisions for therapeutic interventions, and thus, reliable biomarkers are pivotal when considering allergen immunotherapy in the context of precision medicine.

T-cell epitope conservation across allergen species is a major determinant of immunogenicity.

BACKGROUND: Patients with pollen allergies are frequently polysensitized. Pollens contain epitopes that are conserved across multiple species. OBJECTIVE: We sought to demonstrate that cross-reactive T cells that recognize conserved epitopes show higher levels of expansion than T cells recognizing monospecific epitopes because of more frequent stimulation. METHOD: RNA was sequenced from 9 pollens, and the reads were assembled de novo into more than 50,000 transcripts. T-cell epitopes from timothy grass (Phleum pratense) were examined for conservation in these transcripts, and this was correlated to their ability to induce T-cell responses. T cells were expanded in vitro with P pratense-derived peptides and tested for cross-reactivity to pollen extracts in ELISpot assays. RESULTS: We found that antigenic proteins are more conserved than nonimmunogenic proteins in P pratense pollen. Additionally, P pratense epitopes that were highly conserved across pollens elicited more T-cell responses in donors with grass allergy than less conserved epitopes. Moreover, conservation of a P pratense peptide at the transcriptomic level correlated with the ability of that peptide to trigger T cells that were cross-reactive with other non-P pratense pollen extracts. CONCLUSION: We found a correlation between conservation of peptides in plant pollens and their T-cell immunogenicity within P pratense, as well as their ability to induce cross-reactive T-cell responses. T cells recognizing conserved epitopes might be more prominent because they can be stimulated by a broader range of pollens and thereby drive polysensitization in allergic donors. We propose that conserved peptides could potentially be used in diagnostic or immunomodulatory approaches that address the issue of polysensitization and target multiple pollen allergies.

Pectate lyase pollen allergens: sensitization profiles and cross-reactivity pattern.

BACKGROUND: Pollen released by allergenic members of the botanically unrelated families of Asteraceae and Cupressaceae represent potent elicitors of respiratory allergies in regions where these plants are present. As main allergen sources the Asteraceae species ragweed and mugwort, as well as the Cupressaceae species, cypress, mountain cedar, and Japanese cedar have been identified. The major allergens of all species belong to the pectate lyase enzyme family. Thus, we thought to investigate cross-reactivity pattern as well as sensitization capacities of pectate lyase pollen allergens in cohorts from distinct geographic regions. METHODS: The clinically relevant pectate lyase pollen allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1 were purified from aqueous pollen extracts, and patients' sensitization pattern of cohorts from Austria, Canada, Italy, and Japan were determined by IgE ELISA and cross-inhibition experiments. Moreover, we performed microarray experiments and established a mouse model of sensitization. RESULTS: In ELISA and ELISA inhibition experiments specific sensitization pattern were discovered for each geographic region, which reflected the natural allergen exposure of the patients. We found significant cross-reactivity within Asteraceae and Cupressaceae pectate lyase pollen allergens, which was however limited between the orders. Animal experiments showed that immunization with Asteraceae allergens mainly induced antibodies reactive within the order, the same was observed for the Cupressaceae allergens. Cross-reactivity between orders was minimal. Moreover, Amb a 1, Art v 6, and Cry j 1 showed in general higher immunogenicity. CONCLUSION: We could cluster pectate lyase allergens in four categories, Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1, respectively, at which each category has the potential to sensitize predisposed individuals. The sensitization pattern of different cohorts correlated with pollen exposure, which should be considered for future allergy diagnosis and therapy.

Ligand binding modulates the structural dynamics and compactness of the major birch pollen allergen.

Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.

Recombinant allergens for pollen immunotherapy.

Specific immunotherapy (IT) represents the only potentially curative therapeutic intervention of allergic diseases capable of suppressing allergy-associated symptoms not only during treatment, but also after its cessation. Presently, IT is performed with allergen extracts, which represent a heterogeneous mixture of allergenic, as well as nonallergenic, compounds of a given allergen source. To overcome many of the problems associated with extract-based IT, strategies based on the use of recombinant allergens or derivatives thereof have been developed. This review focuses on recombinant technologies to produce allergy therapeuticals, especially for allergies caused by tree, grass and weed pollen, as they are among the most prevalent allergic disorders affecting the population of industrialized societies. The reduction of IgE-binding of recombinant allergen derivatives appears to be mandatory to increase the safety profile of vaccine candidates. Moreover, increased immunogenicity is expected to reduce the dosage regimes of the presently cumbersome treatment. In this regard, it has been convincingly demonstrated in animal models that hypoallergenic molecules can be engineered to harbor inherent antiallergenic immunologic properties. Thus, strategies to modulate the allergenic and immunogenic properties of recombinant allergens will be discussed in detail. In recent years, several successful clinical studies using recombinant wild-type or hypoallergens as active ingredients have been published and, currently, novel treatment forms with higher safety and efficacy profiles are under investigation in clinical trials. These recent developments are summarized and discussed.

Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1.

The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood. Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1. The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.

Response to Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen.

The authors of J. Immunol. 184, 725-735 respond to the article by Rupp (2012), Acta Cryst. F68, 366-376.

Naturally processed T cell-activating peptides of the major birch pollen allergen.

BACKGROUND: Although antigen processing and presentation of allergens to CD4(+)T lymphocytes are key events in the pathophysiology of allergic disorders, they still remain poorly understood. OBJECTIVE: To investigate allergen processing and presentation by dendritic cells using the major birch pollen allergen Bet v 1 as a model. METHODS: Endolysosomal extracts of dendritic cells derived from patients with birch pollen allergy were used to digest Bet v 1. Dendritic cells were pulsed with Bet v 1, and peptides were eluted from MHC class II molecules. Peptides obtained by either approach were sequenced by tandem mass spectrometry. Bet v 1-specific T-cell cultures were stimulated with HLA-DR-eluted Bet v 1-derived peptides. Bet v 1-specific T-cell lines were generated from each patient and analyzed for epitope recognition. RESULTS: A high proportion of Bet v 1 remained intact for a long period of endolysosomal degradation. The peptides that appeared early in the degradation process contained frequently recognized T-cell epitopes. Bet v 1-derived peptides eluted from MHC class II molecules corresponded to those generated by endolysosomal degradation, matched known T-cell epitopes, and showed T cell-activating capacity. The Bet v 1-specific T-cell line of each individual harbored T cells reactive with peptides located within the MHC class II-eluted Bet v 1-derived sequences demonstrating their occurrence in vivo. CONCLUSION: We report for the first time how epitopes of allergens are generated and selected for presentation to T lymphocytes. The limited susceptibility of Bet v 1 to endolysosomal processing might contribute to its high allergenic potential.

Differential T-cell responses and allergen uptake after exposure of dendritic cells to the birch pollen allergens Bet v 1.0101, Bet v 1.0401 and Bet v 1.1001.

The major birch pollen allergen Bet v 1 is present in pollen as a mixture of at least 14 isoforms that share high sequence and structural identities. These isoforms possess either a high or a low IgE-binding capacity which defines them as allergenic or hypoallergenic. Recently, we could demonstrate that only the allergenic isoform Bet v 1.0101 was able to induce an IgE response in birch pollen allergic individuals. The hypoallergenic isoforms Bet v 1.0401 and Bet v 1.1001 were unable to induce IgE synthesis. T-helper cell responses against allergens are characterised by increased levels of Th2 cytokines. Therefore, we examined extent and polarisation of the Th cell response and the kinetics of the allergen uptake after exposure of dendritic cells (DCs) to these isoforms. Monocyte-derived DCs (MDDCs) from birch pollen allergic and non-atopic individuals stimulated with Bet v 1.0101, Bet v 1.0401 or Bet v 1.1001 in combination with the maturation factors TNF-α and IL-1ß resulted in a mature DC phenotype as measured by costimulatory molecule up-regulation. Only Bet v 1.0101-stimulated MDDCs from allergic subjects enhanced proliferation of autologous Th cells and the expression of the Th2 cytokines IL-5 and IL-13. Immature MDDCs of allergic individuals internalised equivalent amounts of the allergenic Bet v 1.0101 and the hypoallergenic Bet v 1.0401. In contrast, the uptake of the hypoallergenic Bet v 1.0401 by immature MDDCs of non-atopic individuals was significantly higher. These results provide evidence that DCs discriminate between allergens and highly related hypoallergens. This process may have an impact on the early phase of sensitisation.

Antigen aggregation decides the fate of the allergic immune response.

Previously, defined naturally occurring isoforms of allergenic proteins were classified as hypoallergens and therefore suggested as an agent for immunotherapy in the future. In this paper, we report for the first time the molecular background of hypoallergenicity by comparing the immunological behavior of hyperallergenic Betula verrucosa major Ag 1a (Bet v 1a) and hypoallergenic Bet v 1d, two isoforms of the major birch pollen allergen Betula verrucosa 1. Despite their cross-reactivity, Bet v 1a and Bet v 1d differ in their capacity to induce protective Ab responses in BALB/c mice. Both isoforms induced similar specific IgE levels, but only Bet v 1d expressed relevant titers of serum IgGs and IgAs. Interestingly, hypoallergenic Bet v 1d activated dendritic cells more efficiently, followed by the production of increased amounts of Th1- as well as Th2-type cytokines. Surprisingly, compared with Bet v 1a, Bet v 1d-immunized mice showed a decreased proliferation of regulatory T cells. Crystallographic studies and dynamic light scattering revealed that Bet v 1d demonstrated a high tendency to form disulfide-linked aggregates due to a serine to cysteine exchange at residue 113. We conclude that aggregation of Bet v 1d triggers the establishment of a protective Ab titer and supports a rationale for Bet v 1d being a promising candidate for specific immunotherapy of birch pollen allergy.

The influence of recombinant production on the immunologic behavior of birch pollen isoallergens.

BACKGROUND: Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity. METHODOLOGY: Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity. CONCLUSION: The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.

Allergy multivaccines created by DNA shuffling of tree pollen allergens.

BACKGROUND: The major allergens of trees belonging to the Fagales order are collectively known as the Bet v 1 family. Members of the Fagales order have distinct geographic distribution, and it is expected that depending on the exposure pattern of the individual, inclusion of other Bet v 1 family members might increase the efficacy of the treatment. OBJECTIVE: We aimed to generate molecules that are suitable for specific immunotherapy not only against birch pollen allergy but also against allergies caused by other cross-reactive tree pollens. METHODS: Fourteen genes of the Bet v 1 family were randomly recombined in vitro by means of DNA shuffling. This library of chimeric proteins was screened for molecules displaying low capacity to induce release of inflammatory mediators but with T-cell immunogenicity higher than that of the parental allergens. RESULTS: Two chimeric proteins were selected from the library of shuffled clones displaying low allergenicity and high immunogenicity, as determined in in vitro assays using human and animal cells and antibodies, as well as in vivo in animal models of allergy. CONCLUSION: Our results show that it is possible to randomly recombine in vitro T- and B-cell epitopes of a family of related allergens and to select chimeric proteins that perfectly match the criteria presently thought to be relevant for improving allergen-specific immunotherapy. CLINICAL IMPLICATIONS: The hypoallergenic chimeras described here recombine epitopes of the major Fagales pollen allergens and thus can efficiently substitute a mixture of extracts used for treating patients with tree pollen-induced spring pollinosis worldwide.