Recombinant Bet Research Articles

IgE to cyclophilins in pollen-allergic children: Epidemiologic, clinical, and diagnostic relevance of a neglected panallergen

Background– Cyclophilins are ubiquitous panallergens whose epidemiological, diagnostic, and clinical relevance is largely unknown and whose sensitization is rarely examined in routine allergy practice. The aim of this study was to investigate the epidemiological, diagnostic, and clinical relevance of cyclophilin in seasonal allergic rhinitis and its comorbidities. Methods– We examined a random sample (25%, n 253) of 1263 Italian children affected by seasonal allergic rhinitis from the “Panallergen in Pediatrics” (PAN-PED) cohort. Patients’ disease phenotype had been already fully characterized through questionnaires (ARIA), skin prick tests (ALK), IgE tests to extracts, major and cross-reactive allergenic molecules of a comprehensive variety of allergenic pollen (immunoCAP), and carbohydrate cross-reacting determinants (CCD) (NOVEOS). We also performed nested studies of sensitization prevalence, correlation and allergen extract inhibition in patients (A) sensitized to birch pollen extract but lacking IgE to Bet v 1, Bet v 2, and Bet v 4, (74/1263) or with the highest serum level of IgE to Bet v 1 (26 within 1263), and (B) in patients with sensitization to extracts of ragweed (18), mugwort (18), pellitory (20), Plantago (19), and plane tree (20), but not to their respective major allergenic molecule, profilins and polcalcins. IgE to cyclophilin was detected with recombinant Bet v 7 (ImmunoCAP) and extract inhibition tests were performed with the same rBet v 7. ResultsIn the randomized population, IgE to rBet v 7 was detected in 43/253 (17%) patients. It was associated with asthma (p<0.028) and oral allergy syndrome (p<0.017) in univariate, but not in a multivariate analysis adjusted for IgE to profilins (Phl p 12), PR.10s (Bet v 1), and LTPs (Pru p 3). IgE to r Bet v 7 was also highly prevalent (47/74; 63%) among patients with unexplained sensitization to birch pollen extract. In patients with unexplained sensitization to ragweed, mugwort, pellitory, Plantago and plane tree pollen, the levels of IgE to those extracts correlated with the levels of IgE to rBet v 7, and they were also significantly inhibited by rBet v 7 (inhibition range 45%-74%). Conclusions– IgE sensitization to cyclophilin is very frequent in pollen-allergic patients living in temperate areas and can produce “false” positive outcomes in SPT and IgE tests to many different pollen extracts. The spectrum of pollen species containing allergenic cyclophilins is probably broader than previously known. Our results suggest that guidelines of molecular diagnostics should include this allergen family and that new tests based on plant cyclophilins should be produced and used with sera of pollen-allergic patients for a more precise identification of the culprit pollen extract and a tailored prescription of allergen immunotherapy.

Natural human Bet v 1-specific IgG antibodies recognize non-conformational epitopes whereas IgE reacts with conformational epitopes.

The nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen-exposed but non-sensitized subjects has not been studied in detail. To investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1-specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1-induced basophil activation. Sera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non-allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG1 and IgG4 reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N-terminal (F1) and C-terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro-array analysis. The ability of Bet v 1-specific serum antibodies from non-allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1-derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non-allergic subjects to modulate Bet v 1-induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients. IgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1 and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen-containing food. Natural Bet v 1-specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1-specific basophil activation. IgE and IgG antibodies from BPA patients and birch pollen-exposed non-sensitized subjects recognize different epitopes. These findings explain why natural allergen-specific IgG do not protect against allergic symptoms and suggest that allergen-specific IgE and IgG have different clonal origin.

Identification of probable pectinesterase as a major allergen of pollen of the Asian white birch (Betula platyphylla) in northern China.

Pollen of the Asian white birch (Betula platyphylla) is a major source of allergens in spring in northern China, yet little research on its pollen allergens has been done so far. To analyze the B. platyphylla pollen allergen profile of patients from northern China and to identify the major pollen allergens in this patient cohort. Sera from 35 Chinese patients with birch pollinosis were collected for this study. The IgE-binding proteins in B. platyphylla pollen extract were analyzed by IgE immunoblots. A novel major allergen was purified by cation exchange chromatography and affinity chromatography. Its IgE reactivity was evaluated by ELISA. The protein was excised from a 2D electrophoresis gel and subjected to ESI-QUAD-TOF. In our study cohort, the prevalence of IgE specific for Bet v 1 was 68.8% (n = 22/32) as measured by ImmunoCAP. In immunoblots, two major bands at around 17 kDa and 70 kDa were detected by IgE from 68.5% (24/35) and 65.7% (23/35) of sera, respectively. The 17 kDa band was identified as Bet v 1 by a monoclonal antibody to Bet v 1 and inhibition experiments with recombinant Bet v 1. The 70 kDa band was a polymorphic glycoprotein and 7 isoforms were found. The protein was identified as probably pectinesterase with a molecular weight of 66 kDa and a PI of 5.7. A 66 kDa protein, probably belonging to pectinesterase family, was identified as a novel major allergen of B. platyphylla pollen in patients from northern China.

Obtaining an expression vector containing the nucleotide sequence of the gene encoding Bet v 1 – the major allergen of birch pollen

An expression vector containing the gene encoding the most common isoform Bet v 1.0101 of the Bet v 1 protein, the major birch pollen allergen, was created for a subsequent expression in the prokaryotic system of Escherichia coli. Total RNA from birch pollen collected in Belarus was used as a matrix. The expression vector pJC40-Веt v 1 was obtained using molecular-genetic methods: cloning, ligation, transformation. The specificity of the cloned fragment was confirmed by sequencing. During the study, 14 rare Escherichia coli codons were identified in the coding part of the cloned gene. The triplets were evenly arranged in the nucleotide sequence; codon clustering was observed only in two cases. The total percentage of rare triplets (8.75 %) and the calculated value of the codon adaptation level (0.57) allow us to predict a sufficiently efficient expression of the studied gene. The data obtained will be used in the synthesis of the recombinant polypeptide Bet v 1.0101.

The Allergen-Specific IgE Concentration Is Important for Optimal Histamine Release From Passively Sensitized Basophils.

BackgroundThe basophil histamine release (HR) assay can be used for allergy diagnosis in addition to the conventional measurement of allergen-specific IgE (sIgE). Passive sensitization of basophils increases the versatility and allows testing the biological relevance of allergen-induced IgE cross-linking in any serum unbiased by the cellular component. However, not all the patient sera perform equally well and we hypothesized that the absolute level and fraction of sIgE affect the performance. Choosing birch pollen allergy as a model, we investigated the concentration of sIgE needed for successful passive sensitization using soluble- or matrix-fixed Bet v 1.MethodsTwenty-eight sera with Bet v 1 sIgE [7 sera within each allergy class (1: 0.1–0.70 kUA/L, 2: 0.71–3.50 kUA/L, 3: 3.51–17.50 kUA/L, and 4+: >17.50 kUA/L)] and a negative control serum pool were used to passively sensitize donor basophils, obtained from buffy coat blood (n = 3). The cells were incubated (30 min) with a soluble allergen (rBet v 1 from 0.2 to 50 ng/ml), matrix-fixed allergen (ImmunoCAP™ containing recombinant Bet v 1), or phorbol 12-myristate 13-acetate (PMA)/ionomycin mixture (maximal HR) and released histamine was quantified fluorometrically.ResultsThe lowest level of Bet v 1 sIgE generating a detectable HR (HR > 10% of maximal release) in all the 3 runs was found to be 1.25 kUA/L (corresponding to allergy class 2, 0.71–3.50 kUA/L). Furthermore, sera from allergy classes 3 and 4+ ascertained a significant reproducible HR: 42/42 vs. 5/21 in allergy class 1 and 15/21 in allergy class 2. Using ImmunoCAP™s containing Bet v 1 as a matrix-fixed allergen system, similar results were obtained where the lowest sIgE concentration mediating an HR was 1.68 kUA/L and 7/7 for both allergy classes 3 and 4+.ConclusionThe results demonstrate that the IgE titer is strikingly robust in predicting the ability to sensitize basophils and produce a measurable HR.

Preventive Administration of Non-Allergenic Bet v 1 Peptides Reduces Allergic Sensitization to Major Birch Pollen Allergen, Bet v 1.

IgE-mediated allergy to birch pollen affects more than 100 million patients world-wide. Bet v 1, a 17 kDa protein is the major allergen in birch pollen responsible for allergic rhinoconjunctivitis and asthma in birch pollen allergic patients. Allergen-specific immunotherapy (AIT) based on therapeutic administration of Bet v 1-containing vaccines is an effective treatment for birch pollen allergy but no allergen-specific forms of prevention are available. We developed a mouse model for IgE sensitization to Bet v 1 based on subcutaneous injection of aluminum-hydroxide adsorbed recombinant Bet v 1 and performed a detailed characterization of the specificities of the IgE, IgG and CD4+ T cell responses in sensitized mice using seven synthetic peptides of 31-42 amino acids length which comprised the Bet v 1 sequence and the epitopes recognized by human CD4+ T cells. We then demonstrate that preventive systemic administration of a mix of synthetic non-allergenic Bet v 1 peptides to 3-4 week old mice significantly reduced allergic immune responses, including IgE, IgG, IgE-mediated basophil activation, CD4+ T cell and IL-4 responses to the complete Bet v 1 allergen but not to the unrelated major grass pollen allergen Phl p 5, without inducing Bet v 1-specific allergic sensitization or adaptive immunity. Our results thus demonstrate that early preventive administration of non-allergenic synthetic T cell epitope-containing allergen peptides could be a safe strategy for the prevention of allergen-specific IgE sensitization.

IgE-cross-blocking antibodies to Fagales following sublingual immunotherapy with recombinant Bet v 1.

Evidence has accumulated that birch pollen immunotherapy reduces rhinoconjunctivitis to pollen of birch homologous trees. Therapeutic efficacy has been associated with IgE-blocking IgG antibodies. We have recently shown that sera collected after 16weeks of sublingual immunotherapy with recombinant Bet v 1 (rBet v 1-SLIT) display strong IgE-blocking bioactivity for Bet v 1. Here, we assessed whether rBet v 1-SLIT-induced IgG antibodies display cross-blocking activity to related allergens in Fagales pollen. IgE, IgG1 and IgG4 reactivity to recombinant Bet v 1, Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1 and Que a 1 were assessed in pre- and post-SLIT samples of 17 individuals by ELISA. A basophil inhibition assay using stripped basophils re-sensitized with a serum pool containing high Bet v 1-specific IgE levels was established and used to assess CD63 expression in response to allergens after incubation with pre-SLIT or post-SLIT samples. IgG1 and IgG4 were depleted from post-SLIT samples to assess its contribution to IgE-cross-blocking. Sublingual immunotherapy with recombinant Bet v 1 boosted cross-reactive IgE antibodies and induced IgG1 and IgG4 antibodies with inter- and intra-individually differing reactivity to the homologs. Highly variable cross-blocking activities of post-SLIT samples to the different allergens were found. IgG1 and IgG4 antibodies displayed cross-blocking activity with individual variance. Our mechanistic approach suggested that immunotherapy with the reference allergen Bet v 1 induces individual repertoires of cross-reactive IgG1 and IgG4 antibodies. The cross-blocking bioactivity of these antibodies was also highly variable and neither predictable from protein homology nor IgE-cross-reactivity.

Development of a sensitive and stable chemiluminescent immunoassay for detection of birch pollen allergic specific IgE based on recombinant Bet v1 protein

BackgroundQuantitative detection of allergens is of great significance for clarifying the cause, treatment, and prevention of allergy disease. Birch pollen is one of the most common inhalational allergens and Bet v1 is the major component allergen of birch allergen. This study aims to develop a stable and sensitive chemiluminescence immunoassay (CLIA) for the detection of birch pollen allergic specific IgE (sIgE) based on recombinant Bet v1 (rBet v1) protein.MethodsrBet v1 protein was expressed in Escherichia coli and purified. Then rBet v1 was applied to detect sIgE in human serum. The performance of the established CLIA was evaluated and compared with Phadia rBet v1 fluorescence enzyme immunoassay (FEIA) system.ResultsThe developed CLIA for sIgE to rBet v1 detection shows excellent performance. The assay showed a linear range from 0.1 to 100 IU/mL, with a low detection limit of 0.06 IU/mL. A total of 164 samples were evaluated by CLIA and compared with the results of FEIA. The positive, negative, and total coincidence rate was 90.6% (87/96), 91.2% (62/68), and 90.9% (149/164), respectively. The r-value of Spearman's rank correlation analysis was 0.935 (P < 0.001). The use of high levels of bilirubin (50 mg/dL), hemoglobin (400 mg/dL) and lipid (2000 mg/dL) didn't interfere with the results.ConclusionsThe proposed CLIA exhibits excellent performance for the detection of rBet v1 specific IgE. It can be a reliable tool for the early diagnosis of hypersensitivity.

Serum immunoglobulin E reactivity to cross-reacting panallergen components in north-eastern Poland patients pollen sensitized

Background: The presence of immunoglobulin E (IgE), which cross-reacts with allergen components, such as profilins, polcalcins, and cross-reacting carbohydrate determinants (CCD), creates a problem when selecting patients for allergen immunotherapy by using conventional methods. The aim of this study was to evaluate the prevalence of sensitization to profilins, polcalcins, and CCDs in patients with seasonal pollen allergic rhinitis. Methods: The study was performed on a group of 112 patients with seasonal pollen allergic rhinitis, ages 14 to 55 years, with sensitization to at least one seasonal allergen (IgE > 0.7 kUA/L). The presence of IgE sensitization to recombinant (r) Bet v 2, rPhl p 12, rBet v 4, rPhl p 7, and CCDs, in addition to rBet v 1, rPhl p 1, rPhl p 5, was evaluated by using a multiparameter immunoblot. Results: Among the studied patients, 64.3, 80.4, and 41.1% were sensitized to birch, timothy grass, and mugwort pollen, respectively. Sensitization to profilins rBet v 2/Phl p 12 was demonstrated in 28.6%, to polcalcins Bet v 4/Phl p 7 in 8.9%, and to CCDs in 25%. In 29.3%, serum IgE reactivity to any of the cross-reactive components could be demonstrated. Serum IgE reactivity to rBet v 2 was always accompanied by IgE reactivity to rPhl p 12, and IgE reactivity to rBet v 4 was always accompanied by IgE reactivity to rPhl p 7. Among the patients with pollinosis co-sensitized to at least two allergen sources according to extract-based diagnosis, possible false-positive results due to sensitization to cross-reactive components were detected in 17.9%. Conclusion: Evaluation of sensitization to cross-reacting components may be useful in evaluation of patients with pollen allergy who are being assessed for allergen immunotherapy to optimize the constitution of their immunotherapy vaccines.

Quality and potency profile of eight recombinant isoallergens, largely mimicking total Bet v 1-specific IgE binding of birch pollen.

To date, only limited information on structure, expression levels and IgE binding of Bet v 1 variants, which are simultaneously expressed in birch pollen, is available. To analyse and compare structure and serum IgE/IgG binding of rBet v 1 variants to Bet v 1.0101. Recombinant Bet v 1 variants were studied with sera of 20 subjects allergic to birch pollen. Folding, aggregation and solubility of the rBet v 1 variants were analysed to attribute diverging IgE binding to either allergen structure or methodological features. IgE/IgG binding was studied with rBet v 1 in solution or adsorbed to solid phases. Allergen-mediated cross-linking of FcεRI receptors was determined by mediator release of sensitized humanized rat basophil leukaemia cells. All variants, except for rBet v 1.0113, were monomeric and had Bet v 1-type conformation. Serum IgE binding to variants adsorbed to solid phase was reduced to 6.6%-36.5% compared with Bet v 1.0101. In contrast, inhibition of IgE binding to Bet v 1.0101 by rBet v 1 variants ranged from 62% to 83%. Similarly, mediator release ranged from 30.7% to 55.2% for all variants and was only clearly reduced for rBet v 1.0301 (10.4%). The IgE-binding potency of rBet v 1 variants representing their native quantities in birch pollen was only slightly lower compared to extract. IgG binding to variants was between 50.9% and 134.5% compared with rBet v 1.0101 (100%). Bet v 1 variants previously classified as hypoallergenic can exhibit similar functional IgE binding as Bet v 1.0101. Eight rBet v 1 variants largely reproduce total Bet v 1-specific IgE binding of birch pollen extracts. Assay format-dependent variation in IgE-binding properties needs to be considered in the development of diagnostic or therapeutic products.

SERUM IGE REACTIVITY TO CROSS-REACTING MOLECULES IN POLLEN ALLERGIC PATIENTS FROM NORTH EASTERN POLAND

Introduction Presence of IgE which cross-react with allergen components such as profilins, polcalcins and cross-reacting carbohydrate determinants (CCD) creates a problem in selection of patients for allergen immunotherapy. This study assesses the prevalence of sensitization to profilins, polcalcins and CCDs in seasonal allergic rhinitis patients (ARs). Methods 112 allergic rhinitis (AR) patients age 14 to 55 years with sensitization to at least one seasonal allergen (IgE>0.7 KU/l) were studied. IgE sensitization to recombinant(r) Bet v 2, rPhl p 12, rBet v 4, rPhl p 7 and CCDs in addition to rBet v 1, rPhl p 1, rPhl p 5 was evaluated using a multiplex immunoblot (Euroimmun, Germany). Results 64.3%, 80.4% and 41.1% of AR patients were sensitized to birch, timothy grass and mugwort pollen, respectively. Sensitization to rBet v 2/Phl p 12 occured in 26.8%, to Bet v 4/Phl p 7 in 8.9%, and to CCDs in 25%. In 29.3% of AR patients serum IgE reactivity to any of the cross-reactive component could be demonstrated. Serum IgE reactivity to rBet v 2 was always accompanied by reactivity to rPhl p 12 and reactivity to rBet v 4 was always accompanied by reactivity to rPhl p 7. Conclusions Evaluation of serum IgE reactivity to cross-reactive components may be useful in assessment of AR patients qualified for allergen immunotherapy.

Determination of Specific Class E Immunoglobulins to Bet v 1 Birch Allergen by the Immuno-PCR Method

The aim of the work is the development of a method of detection of specific class E immunoglobulins (IgE) to the main Bet v 1 birch allergen based on immuno-PCR (iPCR). The recombinant Bet v 1 allergen was obtained in E. coli cells. Its ability to bind to specific IgE was confirmed by enzyme-linked immunosorbent assay (ELISA) using previously characterized sera of individuals with an allergic reaction to birch pollen and control sera in individuals, in which the reaction to this allergen is absent. Based on the obtained recombinant protein, the method of iPCR analysis of specific IgE to Bet v 1 was developed. It was demonstrated that iPCR sensitivity is comparable to ELISA sensitivity, and the titration curves of specific sera in iPCR (unlike those in ELISA) demonstrate a linear dependence; this makes the developed method preferable for quantitative estimation of specific IgE in sera as compared with ELISA.

Unbiased Quantitative Proteomics Reveals a Crucial Role of the Allergen Context for the Activation of Human Dendritic Cells

Worldwide, more than 1 billion people suffer from allergic diseases. However, until now it is not fully understood how certain proteins can induce allergic immune responses, while others cannot. Studies suggest that allergenicity is a process not only determined by properties of the allergen itself but also by costimulatory factors, that are not classically associated with allergic reactions. To investigate the allergenicity of the major birch pollen allergen Bet v 1 and the impact of adjuvants associated with pollen, e.g. lipopolysaccharide (LPS), we performed quantitative proteome analysis to study the activation of monocyte-derived dendritic cells (moDCs). Thus, we treated cells with birch pollen extract (BPE), recombinant Bet v 1, and LPS followed by proteomic profiling via high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) using isobaric labelling. Enrichment and pathway analysis revealed the influence of regulated proteins especially in cytokine signalling and dendritic cell activation. We found highly regulated, but differentially expressed proteins after treatment with BPE and LPS, whereas the cellular response to Bet v 1 was limited. Our findings lead to the conclusion that Bet v 1 needs a specific “allergen context” involving cofactors apart from LPS to induce an immune response in human moDCs.

Endolysosomal protease susceptibility of Amb a 1 as a determinant of allergenicity

summary In the absence of extrinsic adjuvants, purified Amb a 1 induced a strong Th2-polarized immune response in mice. This high allergenic activity seems to be linked to the allergen’s susceptibility to endolysosomal proteolysis.

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.

IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen.

Allergen-specific immunotherapy (AIT) with birch pollen generates Bet v 1-specific immunoglobulin (Ig)G4 which blocks IgE-mediated hypersensitivity mechanisms. Whether IgG4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG4 antibodies are developed is under debate. We sought to analyze the epitope specificities of IgE and IgG4 antibodies from sera of patients who received AIT. 15 sera of patients (13/15 received AIT) with Bet v 1a-specific IgE and IgG4 were analyzed. The structural arrangements of recombinant (r)Bet v 1a and rBet v 1a_11x , modified in five potential epitopes, were analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. IgE binding to Bet v 1 was assessed by ELISA and mediator release assays. Competitive binding of monoclonal antibodies specific for Bet v 1a and serum IgE/IgG4 to rBet v 1a and serum antibody binding to a non-allergenic Bet v 1-type model protein presenting an individual epitope for IgE was analyzed in ELISA and western blot. rBet v 1a_11x had a Bet v 1a - similar secondary and tertiary structure. Monomeric dispersion of rBet v 1a_11x was concentration and buffer-dependent. Up to 1500-fold increase in the EC50 for IgE-mediated mediator release induced by rBet v 1a_11x was determined. The reduction of IgE and IgG4 binding to rBet v 1a_11x was comparable in 67% (10/15) of sera. Bet v 1a-specific monoclonal antibodies inhibited binding of serum IgE and IgG4 to 66.1% and 64.9%, respectively. Serum IgE and IgG4 bound specifically to an individual epitope presented by our model protein in 33% (5/15) of sera. Patients receiving AIT develop Bet v 1a-specific IgG4 which competes with IgE for partly identical or largely overlapping epitopes. The similarities of epitopes for IgE and IgG4 might stimulate the development of epitope-specific diagnostics and therapeutics.

Heat-labile Escherichia coli toxin enhances the induction of allergen-specific IgG antibodies in epicutaneous patch vaccination.

Epicutaneous allergen‐specific immunotherapy (EPIT) is proposed as an alternative route for allergen‐specific immunotherapy (AIT). The induction of allergen‐specific blocking IgG antibodies represents an important mechanism underlying AIT, but has not been investigated for EPIT. Here, we compared the induction of allergen‐specific blocking IgG in outbred guinea pigs which had been immunized with recombinant birch pollen allergen Bet v 1 using patch delivery system (PDS) with or without heat‐labile toxin (LT) from Escherichia coli or subcutaneously with aluminum hydroxide (Alum)‐adsorbed rBet v 1. Only subcutaneous immunization with Alum‐adsorbed rBet v 1 and epicutaneous administration of rBet v 1 with PDS in combination with LT from E. coli induced allergen‐specific IgG antibodies blocking allergic patients' IgE, but not immunization with rBet v 1 via PDS alone. Our results suggest that patch vaccination with rBet v 1 in combination with LT may be a promising strategy for allergen‐specific immunotherapy against birch pollen allergy.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Pimecrolimus Is a Potent Inhibitor of Allergic Reactions to Hymenopteran Venom Extracts and Birch Pollen Allergen In Vitro.

Pimecrolimus (Elidel, SDZ ASM 981) is an anti-inflammatory and immunomodulatory 33-epichloro-derivative of macrolactam ascomycin, with low potential for affecting systemic immune responses compared with other calcineurin inhibitors, cyclosporin A and tacrolimus. Despite numerous studies focused on the mechanism of pimecrolimus action on mast cells, only the single report has addressed pimecrolimus effects on other typical FcεRI-expressing cells, the basophils. Patients allergic to birch pollen (n = 20), hymenopteran venoms (n = 23) and 10 non-allergic volunteers were examined. Primary human basophils pre-treated or not with 0.5–50 μMol pimecrolimus were exposed to various concentrations of recombinant Bet v 1a allergen, bee or wasp venom extracts and anti-IgE for 20 min, and then examined for the expression of CD45, CD193, CD203c, CD63 and CD164 using flow cytometry. The externalization of basophil activation markers (CD63 and CD164) was equally inhibited through pimecrolimus in cells activated by recombinant pollen allergen, hymenopteran venom extracts and anti-IgE. Although the individual response rate was subject to strong variation, importantly, pre-treatment with pimecrolimus lowered the number of activated basophils in response to any of the stimuli in the basophils from all patients. The inhibition was concentration-dependent; approximately half of the basophils were inhibited in the presence of 2.5 mMol pimecrolimus. Pimecrolimus is a valuable new tool for the inhibition of hyper-reactive basophils in patients with pollen allergy and a history of anaphylactic reactions to bee or wasp venoms. Further research should address short-term use of pimecrolimus in vivo in a wide spectrum of allergic diseases.

Frequent occurrence of T cell–mediated late reactions revealed by atopy patch testing with hypoallergenic rBet v 1 fragments

BackgroundLate allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity.ObjectiveWe sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope–containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD).MethodsA clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments. T-cell, cutaneous lymphocyte antigen (CLA)+ and CCR4+ T-cell and cytokine responses were studied by thymidine uptake, carboxyfluorescein diacetate succinimidyl ester staining, and Luminex technology, respectively.ResultsrBet v 1 and hypoallergenic rBet v 1 fragments induced APT reactions in not only most of the patients with birch pollen allergy with AD (11/15) but also in most of those without AD (4/5). Patients with birch pollen allergy with AD had higher Bet v 1–specific proliferation of CLA+ and CCR4+ T cells compared with patients with birch pollen allergy without AD. There were no differences in Bet v 1–specific CLA+ and CCR4+ proliferation and cytokine secretion in patients with and without APT reactions.ConclusionHypoallergenic rBet v 1 fragments induce T cell–dependent late reactions not only in patients with birch pollen allergy with AD but also in those without AD, which can be determined based on APT results but not based on in vitro parameters.