The nanotopography of SiO2 particles impacts the selectivity and 3D fold of bound allergens.

A detailed description of the changes that occur during the formation of protein corona represents a fundamental question in nanoscience, given that it not only impacts the behaviour of nanoparticles but also affects the bound proteins. Relevant questions include whether proteins selectively bind particles, whether a specific orientation is preferred for binding, and whether particle binding leads to a modulation of their 3D fold. For allergens, it is important to answer these questions given that all these effects can modify the allergenic response of atopic individuals. These potential impacts on the bound allergen are closely related to the specific properties of the involved nanoparticles. One important property influencing the formation of protein corona is the nanotopography of the particles. Herein, we studied the effect of nanoparticle porosity on allergen binding using mesoporous and non-porous SiO2 NPs. We investigated (i) the selectivity of allergen binding from a mixture such as crude pollen extract, (ii) whether allergen binding results in a preferred orientation, (iii) the influence of binding on the conformation of the allergen, and (iv) how the binding affects the allergenic response. Nanotopography was found to play a major role in the formation of protein corona, impacting the physicochemical and biological properties of the NP-bound allergen. The porosity of the surface of the SiO2 nanoparticles resulted in a higher binding capacity with pronounced selectivity for (preferentially) binding the major birch pollen allergen Bet v 1. Furthermore, the binding of Bet v 1 to the mesoporous rather than the non-porous SiO2 nanoparticles influenced the 3D fold of the protein, resulting in at least partial unfolding. Consequently, this conformational change influenced the allergenic response, as observed by mediator release assays employing the sera of patients and immune effector cells. For an in-depth understanding of the bio-nano interactions, the properties of the particles need to be considered not only regarding the identity and morphology of the material, but also their nanotopography, given that porosity may greatly influence the structure, and hence the biological behaviour of the bound proteins. Thus, thorough structural investigations upon the formation of protein corona are important when considering immunological outcomes, as particle binding can influence the allergenic response elicited by the bound allergen.

Purification and biochemical characterization of Hel a 6, a cross-reactive pectate lyase allergen from Sunflower (Helianthus annuus L.) pollen.

Sunflower pollen was reported to contain respiratory allergens responsible for occupational allergy and pollinosis. The present study describes the comprehensive characterization of a major sunflower allergen Hel a 6. Natural Hel a 6 was purified from sunflower pollen by anion exchange and gel filtration chromatography. Hel a 6 reacted with IgE-antibodies from 57% of 39 sunflower-sensitized patient sera suggesting it to be a major allergen. The patients were of Indian origin and suffering from pollinosis and allergic rhinitis. Hel a 6 exhibited allergenic activity by stimulating mediator release from basophils. Monomeric Hel a 6 displayed pectate lyase activity. The effect of various physicochemical parameters such as temperature, pH, and calcium ion on the functional activity of Hel a 6 revealed a stable nature of the protein. Hel a 6 was folded, and its melting curve showed reversible denaturation in which it refolded back to its native conformation from a denatured state. Hel a 6 displayed a high degree of sequence conservation with the pectate lyase allergens from related taxonomic families such as Amb a 1 (67%) and Art v 6 (57%). The IgE-cross reactivity was observed between Hel a 6 and its ragweed and mugwort homologs. The cross-reactivity was further substantiated by the mediator release when Hel a 6-sensitized effector cells were cross-stimulated with Art v 6 and Amb a 1. Several putative B cell epitopes were predicted and mapped on these 3 allergens. Two antigenic regions were found to be commonly shared by these 3 allergens, which could be crucial for cross-reactivity. In conclusion, Hel a 6 serves as a candidate molecule for diagnosis and immunotherapy for weed allergy.

Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

An indirect comparison meta-analysis of AS03 and MF59 adjuvants in pandemic influenza A(H1N1)pdm09 vaccines.

BACKGROUND: Although oil-in-water adjuvants improve pandemic influenza vaccine efficacy, AS03 versus MF59 adjuvant comparisons in A(H1N1)pdm09 pandemic vaccines are lacking. METHODS: We conducted an indirect-comparison meta-analysis extracting published data from randomised controlled trials in literature databases (01/01/2009-09/09/2018), evaluating immunogenicity and safety of AS03- or MF59-adjuvanted vaccines. We conducted comparisons of log-transformed haemagglutination inhibition geometric mean titre ratio (GMTR; primary outcome) of different regimens of each adjuvant versus unadjuvanted counterparts. Then via test of subgroup differences, we indirectly compared different AS03 versus MF59 regimens. RESULTS: We identified 22 publications with 10,734 participants. In adults, AS03-adjuvanted vaccines (3.75 µg haemagglutinin) achieved superior GMTR versus unadjuvanted vaccines (all four comparisons); MD = 0.56 (95%CI 0.33 to 0.80, p < 0.001) to 1.18 (95%CI 0.72 to 1.65, p < 0.001). MF59 (full-dose)-adjuvanted vaccines (7.5 µg haemagglutinin) were superior to unadjuvanted vaccines (three of four comparisons); MD = 0.47 (95%CI 0.19 to 0.75, p = 0.001) to 0.80 (95%CI 0.44 to 1.16, p < 0.001). Adult indirect comparisons favoured AS03 over MF59 (six of eight comparisons; p < 0.001 to p = 0.088). Paediatric indirect comparisons favoured MF59-adjuvanted vaccines (two of seven comparisons; p = 0.011, 0.079). However, unadjuvanted control group seroconversion rate was lower in MF59 than AS03 studies (p < 0.001 to p = 0.097). There was substantial heterogeneity, and adult AS03 studies had lower risk of bias. CONCLUSIONS: Despite limited studies, in adults, AS03-adjuvanted vaccines allow antigen sparing versus MF59-adjuvanted and unadjuvanted vaccines, with similar immunogenicity, but higher risk of pain and fatigue (secondary outcomes) than unadjuvanted vaccines. In children, adjuvanted vaccines are also superior, but the better adjuvant is uncertain.

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.

Fusion proteins of flagellin and the major birch pollen allergen Bet v 1 show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity.

BACKGROUND: Recombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins can enhance the efficacy of allergen-specific immunotherapy. OBJECTIVE: We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines. METHODS: A truncated version of flagellin (NtCFlg) was genetically fused to the N- or C-terminus of Bet v 1. Toll-like receptor (TLR) 5 binding was assessed with HEK293 cells expressing TLR5. Upregulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells from allergic patients was analyzed by using flow cytometry. The T cell-stimulatory capacity of the fusion proteins was assessed with naive and Bet v 1-specific T cells. IgE binding was tested in inhibition ELISAs and basophil activation tests. Mice were immunized with the fusion proteins in the absence and presence of aluminum hydroxide. Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in basophil activation tests. RESULTS: Both fusion proteins matured monocyte-derived dendritic cells through TLR5. Compared with Bet v 1, the fusion proteins showed stronger T cell-stimulatory and reduced IgE-binding capacity and induced murine Bet v 1-specific antibodies in the absence of aluminum hydroxide. However, only antibodies induced by means of immunization with NtCFlg fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1. CONCLUSION: Bet v 1-flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity and thus represent promising vaccines for birch pollen allergen-specific immunotherapy. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunologic characteristics.

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.

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.

Osteogenesis imperfecta and primary open angle glaucoma: genotypic analysis of a new phenotypic association.

PURPOSE: Osteogenesis imperfecta (OI) is a group of inherited disorders characterized by bone fragility. Ocular findings include blue sclera, low ocular rigidity, and thin corneal thickness. However, there are no documented cases linking OI and primary open angle glaucoma (POAG). In this report, we describe three individuals, one isolated case and two from a multiplex family, with OI type I and POAG. METHODS: Available family members with OI and POAG had a complete eye examination, including visual acuity, intraocular pressure (IOP), pachymetry, slit-lamp exam, dilated fundus exam, and visual fields. DNA from blood samples was sequenced and screened for mutations in COL1A1/2 and myocilin (MYOC). RESULTS: All subjects had OI type I. Findings of POAG included elevated IOP, normal gonioscopy, and glaucomatous optic disc cupping and visual field loss. POAG cosegregated with OI in the multiplex family. The multiplex family had a single nucleotide insertion (c.540_541insC) in COL1A1 resulting in a frameshift mutation and a premature termination codon. The sporadic case had a COL1A1 splice acceptor site mutation (c.2452-2A>T or IVS36-2A>T) predicted to result in a premature termination codon due to intron inclusion or a cryptic splice site. None of the glaucoma cases had mutations or sequence changes in MYOC. CONCLUSIONS: We identified two novel mutations in COL1A1 in individuals with OI type I and POAG. Thus, some mutations in COL1A1 may be causative for OI and POAG. Alternatively, susceptibility genes may interact with mutations in COL1A1 to cause POAG.

Meta-analysis of genome-wide association studies identifies novel loci that influence cupping and the glaucomatous process.

Glaucoma is characterized by irreversible optic nerve degeneration and is the most frequent cause of irreversible blindness worldwide. Here, the International Glaucoma Genetics Consortium conducts a meta-analysis of genome-wide association studies of vertical cup-disc ratio (VCDR), an important disease-related optic nerve parameter. In 21,094 individuals of European ancestry and 6,784 individuals of Asian ancestry, we identify 10 new loci associated with variation in VCDR. In a separate risk-score analysis of five case-control studies, Caucasians in the highest quintile have a 2.5-fold increased risk of primary open-angle glaucoma as compared with those in the lowest quintile. This study has more than doubled the known loci associated with optic disc cupping and will allow greater understanding of mechanisms involved in this common blinding condition.

Allergens of weed pollen: an overview on recombinant and natural molecules.

Weeds represent a botanically unrelated group of plants that usually lack commercial or aesthetical value. Pollen of allergenic weeds are able to trigger type I reactions in allergic patients and can be found in the plant families of Asteraceae, Amaranthaceae, Plantaginaceae, Urticaceae, and Euphorbiaceae. To date, 34 weed pollen allergens are listed in the IUIS allergen nomenclature database, which were physicochemically and immunologically characterized to varying degrees. Relevant allergens of weeds belong to the pectate lyase family, defensin-like family, Ole e 1-like family, non-specific lipid transfer protein 1 family and the pan-allergens profilin and polcalcins. This review provides an overview on weed pollen allergens primarily focusing on the molecular level. In particular, the characteristics and properties of purified recombinant allergens and hypoallergenic derivatives are described and their potential use in diagnosis and therapy of weed pollen allergy is discussed.

Molecular and immunological characterization of ragweed (Ambrosia artemisiifolia L.) pollen after exposure of the plants to elevated ozone over a whole growing season.

Climate change and air pollution, including ozone is known to affect plants and might also influence the ragweed pollen, known to carry strong allergens. We compared the transcriptome of ragweed pollen produced under ambient and elevated ozone by 454-sequencing. An enzyme-linked immunosorbent assay (ELISA) was carried out for the major ragweed allergen Amb a 1. Pollen surface was examined by scanning electron microscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and phenolics were analysed by high-performance liquid chromatography. Elevated ozone had no influence on the pollen size, shape, surface structure or amount of phenolics. ATR-FTIR indicated increased pectin-like material in the exine. Transcriptomic analyses showed changes in expressed-sequence tags (ESTs), including allergens. However, ELISA indicated no significantly increased amounts of Amb a 1 under elevated ozone concentrations. The data highlight a direct influence of ozone on the exine components and transcript level of allergens. As the total protein amount of Amb a 1 was not altered, a direct correlation to an increased risk to human health could not be derived. Additional, the 454-sequencing contributes to the identification of stress-related transcripts in mature pollen that could be grouped into distinct gene ontology terms.

Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals.

T cells play an important role in the pathogenesis of allergic diseases. However, the proteins considered as potential immunogens of allergenic T-cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is a well-studied inhaled allergen for which major IgE-reactive allergens have also been shown to trigger T helper 2 (Th2) responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE reactivity. A TG pollen extract was analyzed by 2D gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry of selected protein spots in combination with de novo sequencing of the whole TG pollen transcriptome identified 93 previously undescribed proteins for further study, 64 of which were not targeted by IgE. Predicted MHC binding peptides from the previoulsy undescribed TG proteins were screened for T-cell reactivity in peripheral blood mononuclear cells from allergic donors. Strong IL-5 production was detected in response to peptides from several of the previously undescribed proteins, most of which were not targeted by IgE. Responses against the dominant undescribed epitopes were associated with the memory T-cell subset and could even be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic, proteomic, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.

Humoral and cellular cross-reactivity between Amb a 1, the major ragweed pollen allergen, and its mugwort homolog Art v 6.

Ragweed and mugwort are closely related weeds that represent the major cause of pollen allergy in late summer. Concomitant sensitization and clinical cross-reactivity frequently occur in subjects who are coexposed to both pollen species, and have implications for diagnosis and specific immunotherapy. Molecules involved in this cross-reactivity might be Amb a 1, the major ragweed pollen allergen, and Art v 6, a highly homologous allergen from mugwort. Therefore, we investigated the IgE and T cell response to Art v 6 of 60 weed pollen-allergic patients and assessed its immunological cross-reactivity with Amb a 1. Results of ELISA inhibition experiments suggested that both allergens are largely cross-reactive, but Amb a 1 possesses more IgE epitopes than Art v 6. In patients with IgE to both allergens, Amb a 1-induced T cell lines and clones responded weakly to Art v 6. Moreover, Art v 6-induced T cell lines responded stronger to Amb a 1. T cell epitope mapping of Art v 6 revealed that it contains only a few cross-reactive epitopes, which is opposed to the multiple T cell-activating regions present in Amb a 1. In summary, Amb a 1 can elicit more diverse allergen-specific IgE and T cell responses than Art v 6 and dominates the cross-reactivity with its homolog. Nevertheless, Art v 6 can act as a primary sensitizing allergen in areas with high mugwort pollen exposure, and consequently may facilitate sensitization to Amb a 1 by epitope cross-recognition of T and B cells.

Sensitization prevalence, antibody cross-reactivity and immunogenic peptide profile of Api g 2, the non-specific lipid transfer protein 1 of celery.

BACKGROUND: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. METHODOLOGY: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. RESULTS: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. CONCLUSIONS: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

The role of lipid transfer proteins in allergic diseases.

Nonspecific lipid transfer proteins (LTPs) are important allergens in fruits, vegetables, nuts, pollen, and latex. Despite their wide distribution throughout the plant kingdom, their clinical relevance is largely confined to the Mediterranean area. As they can sensitize via the gastrointestinal tract, LPTs are considered true food allergens, and IgE reactivity to LTPs is often associated with severe systemic symptoms. Although Pru p 3 represents the predominant LTP in terms of patients' IgE recognition, the contribution of pollen LTPs in primary sensitization cannot be ruled out. Due to structural homology, LTPs from different allergen sources are generally IgE cross-reactive. However, sensitization profiles among allergic patients are extremely heterogeneous, and individual cross-reactivity patterns can be restricted to a single LTP or encompass many different LTPs. Molecule-based approaches in allergy research and diagnosis are important for better understanding of LTP allergy and could assist clinicians with providing adequate patient-tailored advice.

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.