Combined analysis of five non-interventional studies of the effectiveness, tolerability, and safety of the extrafine fixed dose beclomethasone/formoterol combination in the treatment of asthma in Austria.

OBJECTIVE: The real-world effectiveness and tolerability of an extrafine fixed dose beclomethasone/formoterol (BDP/FF) treatment of patients with partially or non-controlled asthma was evaluated in five non-interventional studies (NISs) from Austria. METHODS: Asthma patients enrolled in these five NISs were treated with beclomethasone/formoterol (Foster® or Foster® Nexthaler®) as maintenance and reliever over 12 weeks. Asthma control, lung function and symptom scores were assessed at baseline, after 4-8 weeks and at the end of the investigations in week 12. In addition, tolerability and handling of the devices were evaluated by questionnaires. RESULTS: The combined analysis included 891 patients (53% female, aged 49.3 years) demonstrating significant improvements in asthma control, lung function parameters (PEF, FEV1 and FVC) and symptom scores (reduction of breathlessness, wheezing, chest tightness and cough). These changes were already detectable after 4-8 weeks. The treatment was effective irrespective of smoking status, exercise, or previous medication. Tolerability of the therapy with extrafine BDP/FF was rated as "very good" or "good" in 98% of the patients. 95% of the patients intended to continue the treatment, and nearly all (99%) rated the handling of the device as "very good" or "good". No serious adverse reactions were reported. CONCLUSIONS: This combined analysis of five non-interventional studies confirms the effectiveness and tolerability of the extrafine fixed-dose BDP/FF combination (Foster® and Foster® Nexthaler®) in a heterogenous patient population suffering from partially or non-controlled asthma. Therapy was associated with a high patient satisfaction and the absence of serious adverse reactions.

Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

BACKGROUND: Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. OBJECTIVE: To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. METHODS: A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. RESULTS: A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. CONCLUSION: Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients.

Intranasal administration of allergen increases specific IgE whereas intranasal omalizumab does not increase serum IgE levels-A pilot study.

BACKGROUND: Administration of the therapeutic anti-IgE antibody omalizumab to patients induces strong increases in IgE antibody levels. OBJECTIVE: To investigate the effect of intranasal administration of major birch pollen allergen Bet v 1, omalizumab or placebo on the levels of total and allergen-specific IgE in patients with birch pollen allergy. METHODS: Based on the fact that intranasal allergen application induces rises of systemic allergen-specific IgE, we performed a double-blind placebo-controlled pilot trial in which birch pollen allergic subjects were challenged intranasally with omalizumab, placebo or birch pollen allergen Bet v 1. Total and allergen-specific IgE, IgG and basophil sensitivity were measured before and 8 weeks after challenge. For control purposes, total, allergen-specific IgE levels and omalizumab-IgE complexes as well as specific IgG levels were studied in subjects treated subcutaneously with either omalizumab or placebo. Effects of omalizumab on IgE production by IL-4/anti-CD40-treated PBMCs from allergic patients were studied in vitro. RESULTS: Intranasal challenge with Bet v 1 induced increases in Bet v 1-specific IgE levels by a median of 59.2%, and this change differed significantly from the other treatment groups (P = .016). No relevant change in allergen-specific and total IgE levels was observed in subjects challenged with omalizumab. Addition of omalizumab did not enhance IL-4/anti-CD40-induced IgE production in vitro. Significant rises in total IgE (mean IgE before: 131.83 kU/L to mean IgE after: 505.23 kU/L) and the presence of IgE-omalizumab complexes were observed after subcutaneous administration of omalizumab. CONCLUSION: Intranasal administration of allergen induced rises of allergen-specific IgE levels, whereas intranasal administration of omalizumab did not enhance systemic total or allergen-specific IgE levels.

Hypoallergenic derivatives of Fel d 1 obtained by rational reassembly for allergy vaccination and tolerance induction.

BACKGROUND AND OBJECTIVE: The major cat allergen Fel d 1 represents one of the most important respiratory allergens. Aim of this study was to engineer recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes for vaccination and tolerance induction. METHODS: Seven recombinant mosaic proteins were generated by reassembly of non-IgE-reactive peptides of Fel d 1 which contained the sequence elements for induction of allergen-specific blocking IgG antibodies and T cell epitopes. Mosaic proteins were expressed in Escherichia coli using codon-optimized synthetic genes and compared with Fel d 1 regarding structural fold by circular dichroism, IgE-binding capacity, activation of allergic patients' basophils and ability to induce allergen-specific blocking IgG antibodies upon immunization. RESULTS: Although each of the mosaic proteins had lost the alpha-helical fold typical for Fel d 1, a strong reduction in IgE reactivity as well as allergenic activity in basophil activation assays was only obtained for three constructs, two reassembled fragments (Fel d 1 MB, Fel d 1 MC) and a fusion of the latter two (Fel d 1 MF) in which the cysteines of Fel d 1 MC were replaced by serines. Immunization of rabbits with Fel d 1 MB, MC and MF induced high levels of IgG antibodies that inhibited IgE reactivity of cat-allergic patients to Fel d 1 in a comparable manner as IgG induced with the wild-type allergen. CONCLUSIONS: We report the development of hypoallergenic reassembled Fel d 1 proteins suitable for vaccination and tolerance induction in cat-allergic patients.

The majority of allergen-specific IgE in the blood of allergic patients does not originate from blood-derived B cells or plasma cells.

BACKGROUND: The production of allergen-specific IgE antibodies is a hallmark of IgE-mediated allergy but the contribution of blood cells to allergen-specific IgE production in allergic patients has not been studied in detail. OBJECTIVE: Aim of this study was the characterization of IgE-producing cells in the blood of allergic patients and the determination of the amount of IgE antibodies which are produced by these cells in relation to total amounts of circulating specific IgE. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from allergic patients and cell populations were purified or depleted using magnetically labelled antibodies directed against specific cell surface markers (CD19, CD20, CD22, CD27, CD38, CD126, CD138, CD203c). Allergen-specific IgE was measured in serum samples and cell culture supernatants by quantitative ImmunoCAP measurements and by ELISA using purified recombinant allergens. IgE transcripts were detected using RT-PCR with primers specific for human IgE. RESULTS: We found that allergen-specific IgE levels in PBMC supernatants correlated strongly with specific serum IgE but represented less than 1% of circulating IgE. Depletion of basophils resulted in substantial reduction of allergen-specific IgE levels in PBMC culture supernatants suggesting that an important source of allergen-specific IgE in PBMC supernatants could be IgE derived from the surface of basophils. Newly synthesized IgE was derived from CD138+ plasma cells, but not from B and B memory cells, and accounted for only approximately 0.2% of circulating IgE in blood. CONCLUSION AND CLINICAL RELEVANCE: Our finding that the majority of allergen-specific IgE in the peripheral blood is not derived from IgE-secreting cells in the blood suggests local IgE production in tissues as a major source for allergen-specific IgE and possible target for therapeutic intervention.

Allergen-specific immunotherapy: from therapeutic vaccines to prophylactic approaches.

Immunoglobulin E-mediated allergies affect more than 25% of the population. Allergen exposure induces a variety of symptoms in allergic patients, which include rhinitis, conjunctivitis, asthma, dermatitis, food allergy and life-threatening systemic anaphylaxis. At present, allergen-specific immunotherapy (SIT), which is based on the administration of the disease-causing allergens, is the only disease-modifying treatment for allergy. Current therapeutic allergy vaccines are still prepared from relatively poorly defined allergen extracts. However, with the availability of the structures of the most common allergen molecules, it has become possible to produce well-defined recombinant and synthetic allergy vaccines that allow specific targeting of the mechanisms of allergic disease. Here we provide a summary of the development and mechanisms of SIT, and then review new forms of therapeutic vaccines that are based on recombinant and synthetic molecules. Finally, we discuss possible allergen-specific strategies for prevention of allergic disease.

Developments in allergen-specific immunotherapy: from allergen extracts to allergy vaccines bypassing allergen-specific immunoglobulin E and T cell reactivity.

Allergen-specific immunotherapy (SIT) is the only specific and disease-modifying approach for the treatment of allergy but several disadvantages have limited its broad applicability. We argue that the majority of the possible disadvantages of SIT such as unwanted effects, poor efficacy and specificity as well as inconvenient application are related to the poor quality of natural allergen extracts, which are the active ingredients of all currently available allergy vaccines. Because of the progress made in the field of molecular allergen characterization, new allergy vaccines based on recombinant allergens, recombinant hypoallergenic allergen derivatives and allergen-derived T cell peptides have entered clinical testing and hold promise to reduce the side-effects and to increase the specificity as well as the efficacy of SIT. Here, we present a refined immunotherapy concept, which is based on the use of peptides derived from allergen surfaces that exhibit reduced, allergen-specific IgE as well as T cell reactivity. These peptides when fused to non-allergenic carriers give rise to allergen-specific protective IgG responses with T cell help from a non-allergenic carrier molecule. We summarize the experimental data demonstrating that such peptide vaccines can bypass allergen-specific IgE as well as T cell activation and may be administered at high doses without IgE- and T cell-mediated side-effects. Should these peptide vaccines prove efficacious and safe in clinical trials, it may become possible to develop convenient, safe and broadly applicable forms of SIT as true alternatives to symptomatic, drug-based allergy treatment.

Molecular composition and biological activity of commercial birch pollen allergen extracts.

BACKGROUND: Commercial extracts used for diagnosis and treatment of allergy are currently prepared from natural allergen sources. The aim of this study was to analyse birch pollen allergen extracts produced for in vivo diagnosis of birch pollen allergy regarding their contents of individual birch pollen allergens (Bet v 1, Bet v 2 and Bet v 4). METHODS: Protein contents were measured and the allergen composition was analysed by immunoblotting using antibody probes specific for Bet v 1, Bet v 2 and Bet v 4 in birch pollen extracts from five manufacturers of allergen extracts. The contents of the major birch pollen allergen, Bet v 1, were quantified with a specific two-site binding enzyme-linked immunosorbent assay with nanogram sensitivity for Bet v 1. The biological activities of the allergen extracts were evaluated by skin prick testing in birch pollen allergic patients and compared with their sensitization profiles. RESULTS: A more than 10-fold variation regarding total protein contents (23.1-314 microg mL(-1)) and also regarding the amounts of the major birch pollen allergen, Bet v 1 (1.62-19.6 microg mL(-1)) was found. The highly cross-reactive Bet v 4 allergen was absent in three of the five tested extracts. Furthermore, varying skin test results were obtained in birch pollen allergic patients with the allergen extracts. CONCLUSIONS: Commercial birch pollen extracts exhibit a considerable variability regarding allergen contents and hence deliver varying in vivo test results. These problems might be overcome with recombinant allergen-based preparations.

Heterogeneity of commercial timothy grass pollen extracts.

BACKGROUND: The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources. OBJECTIVE: To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity. METHODS: Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients. RESULTS: The allergen extracts showed broad variations in protein compositions and amounts (24.1-197.7 microg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32-384 ng/mL; Phl p 2: 1128-6530 ng/mL, Phl p 5: 40-793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients. CONCLUSIONS: Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.

Recombinant allergens promote expression of aminopeptidase-n (CD13) on basophils in allergic patients.

IgE-dependent activation of basophils is associated with upregulation of several surface molecules. We recently identified the surface enzyme aminopeptidase N (CD13) as a novel activation antigen on human basophils. In the present study, we asked whether CD13 can be employed as a novel marker of allergen-induced activation of basophils in allergic individuals. Patients allergic to major allergens from grass pollen (Phl p 1, Phl p 5), birch pollen (Bet v 1), or house dust mites (Der p 2), were examined. Blood basophils were exposed to various concentrations of recombinant allergens for 15 minutes, and examined for expression of CD13 by multicolor flow cytometry. The allergen-induced upregulation of CD13 was compared with allergen-dependent increases in expression of CD63 and CD203c. Exposure to recombinant allergens resulted in an increase in expression of CD13 on basophils in all sensitized individuals, whereas no increase in CD13 was seen in healthy controls. The effects of the recombinant allergens on CD13-expression were dose- and time-dependent, were not observed in the absence of extracellular calcium, and were counteracted by preincubation of basophils with the PI3-kinase-targeting drugs staurosporin and LY294002. There was a good correlation between allergen-induced upregulation of CD13, CD63, and CD203c on basophils. In aggregate, our data show that recombinant allergens promote expression of CD13 on basophils in sensitized individuals. The functional significance and diagnostic implications of this observation remain to be determined.