Sublingual immunotherapy: from biological extracts to recombinant allergens

Sublingual vaccines based on biological extracts from various natural allergen sources are effective in the treatment of respiratory allergies. These vaccines comprise a complex mixture of proteins and glycoproteins that require dedicated standardization procedures to ensure batch-to-batch consistency. Because of the lack of correlation between the potency of an allergen extract and the quantity of major allergen content, standardization is achieved predominantly by determining the global IgE binding capacity of the extract in vitro . New proteomic technologies can be used to further characterize the most abundant proteins present in an extract. Second-generation sublingual vaccines based on recombinant allergens are under development. The aim is to produce molecularly defined vaccines that exhibit superior efficacy, while allowing for simplified immunization schedules. In this approach, recombinant DNA technology is used to express highly purified allergens in their native (i.e. wild-type) conformation. The recombinant allergens are then formulated with ad hoc adjuvants and/or mucoadhesive galenic excipients so that they specifically target oral Langerhans cells and induce allergen-specific regulatory T cells.     

The structural basis of allergenicity: recombinant DNA-based strategies for the study of allergens

The use of recombinant DNA techniques for the study of allergenicity of proteins is a viable, and in many ways a preferred, alternative to the traditional procedures of protein purification, digestion and analysis of peptides for both allergenicity and amino acid sequence. The process of protein purification can be difficult and in many instances workers are forced to use only partially pure fractions that make the identification of the allergenic proteins uncertain. Furthermore, the purification and sequencing of peptides and their testing for retention of allergenic properties, represents a substantial and time-consuming work load. The synthesis of families of synthetic peptides to characterize the amino acids important for allergenic properties is also expensive and time-consuming. On the other hand, the preparation of a cDNA library from an allergen source is today a relatively easy and inexpensive task. The isolation and purification of cDNA clones is comparatively trivial compared to protein purification. Using the techniques described in this text, it can be seen that the molecular biological approach, although in some respects similar in principle to those of the protein chemist to study allergens, provides the capability to study several clones at the same time, and to compare clones for the presence of conserved regions corresponding to allergenic determinants. In addition, the techniques for generating mutant sequences provides perhaps the most powerful and simple set of procedures available for defining the amino acid structures essential for proteins or peptides to behave as allergens.     

Recombinant allergens for allergen-specific immunotherapy: 10 years anniversary of immunotherapy with recombinant allergens

The broad applicability of allergen-specific immunotherapy for the treatment and eventually prevention of IgE-mediated allergy is limited by the poor quality and allergenic activity of natural allergen extracts that are used for the production of current allergy vaccines. Today, the genetic code of the most important allergens has been deciphered; recombinant allergens equalling their natural counterparts have been produced for diagnosis and immunotherapy, and a large panel of genetically modified allergens with reduced allergenic activity has been characterized to improve safety of immunotherapy and explore allergen-specific prevention strategies. Successful immunotherapy studies have been performed with recombinant allergens and hypoallergenic allergen derivatives and will lead to the registration of the first recombinant allergen-based vaccines in the near future. There is no doubt that recombinant allergen-based vaccination strategies will be generally applicable to most allergen sources, including respiratory, food and venom allergens and allow to produce safe allergy vaccines for the treatment of the most common forms of IgE-mediated allergies.     

Rapid method for arrayed investigation of IgE-reactivity profiles using natural and recombinant allergens

BACKGROUND: The availability of increasing numbers of purified natural and recombinant allergens offer the possibility for component-resolved characterization of IgE binding. To make use of this potential, fast and simple methods with high capacity have to be developed. METHODS: A laboratory multiscreen device was used in an innovative two-dimensional approach. In the first step, natural and recombinant allergens were immobilized onto the membrane using the sample chambers as application mask and, after blocking and rotating the membrane through 90 degrees, the same device was used to apply and incubate sera of allergic patients. Enzyme-linked immunoassay (ELISA) quantification of specific IgE was performed for purposes of comparison. RESULTS: Proteins were most efficiently bound onto nitrocellulose in 20 mM sodium hydroxide (NaOH). Up to 45 proteins or extracts could be investigated with a maximum of 45 sera in a single application, resulting in a resolution of 2,025 spots on one membrane with a size comparable to a standard Western blot. A high correlation for IgE-binding between natural and recombinant allergens was observed. Development of the membrane resulted in very evenly distributed square patterns. The results corresponded with the conventional ELISA measurements of specific IgE. CONCLUSIONS: The innovative usage of a standard incubation device for both application of proteins as well as screening of sera provides a simple high throughput method for the characterization of IgE binding to allergens. The results are important for component resolved diagnosis of allergy by means of fast monitoring of IgE- and IgG-reactivity spectra. Recombinant allergens may be used as targets for these purposes.     

Antibody profiles and self-reported symptoms to pollen-related food allergens in grass pollen-allergic patients from northern Europe

BACKGROUND: Most studies on pollen-related food allergy have so far focused on the association of birch/weed pollen allergens and plant food allergy. The aim of this study was to elucidate the allergen spectrum among a group of grass pollen-allergic patients from northern Europe and to relate the results to clinical histories of pollen-related food allergy. METHODS: Fifty-eight grass pollen-allergic patients answered a questionnaire regarding allergy to foods. Blood samples were taken to test IgE-reactivity to a large panel of pollen allergens and pollen- and nonpollen-related food allergens using crude allergen extracts and recombinant and native allergens. RESULTS: Three different groups of grass pollen-allergic patients were identified according to their IgE antibody profile: a grass pollen group only (19%), a grass and tree pollen group (29%) and a grass, tree and compositae (pan-) pollen group (48%). No sensitization to Bet v 1 as well as almost no IgE to plant food was observed in the grass pollen group. In contrast, nearly all patients in the two tree-related groups had IgE to Bet v 1, which reflected the high frequency of adverse reactions to typical birch-related food in these groups. Only four patients belonging to the pan-pollen group displayed IgE to profilin Phl p 12/Bet v 2. Patients in the pan-pollen group reported significantly more symptoms to food allergens compared with patients in the two other groups. The most frequently reported symptom was the oral allergy syndrome. CONCLUSIONS: Sensitization to grass pollen alone is rare among grass pollen-allergic patients from northern Europe. The majority of patients are in addition sensitized to birch (Bet v 1), which seems to be closely related to their pollen-derived food allergy. The study highlights the advantage of using well-defined allergen molecules for the diagnosis of cross-reactivity between pollen and food allergens.     

Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E

BACKGROUND: The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen-specific IgE. OBJECTIVE: To test the performance of this allergen microarray in a serological analytical study. METHODS: Standard allergens contained in grass pollen (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) and tree pollen (Bet v 1 and Bet v 2) were used as a model system. The detection of allergen-specific serum IgE using microarrays was compared with standard test systems: CAP/RAST and an in-house ELISA. In order to test the analytical sensitivity of the assays, geometric dilutions of a serum pool containing high levels of pollen-specific IgE from allergic individuals were tested in each system. To assess the analytical specificity, the sera of 51 patients with presumptive allergic symptoms were collected before diagnosis. Thereafter, the results for grass/tree-pollen-specific IgE were compared. RESULTS: The microarray has a good dynamic range similar to the CAP/RAST system. Microarray and ELISA showed comparable analytical sensitivity exceeding the CAP/RAST system. With respect to the analytical specificity, no significant cross-reactivity of the allergens was observed. For two of the allergens tested, weak positive signals were detected in the microarray test system, whereas they were not detectable by CAP/RAST. CONCLUSION: A good correlation of presently used methods to detect serum IgE and the novel microarray test system was observed. As a next step, a careful validation of this method for a multitude of allergens and a thorough clinical evaluation has to be provided. Microarray testing of allergen-specific IgE can be presumed to be the method of choice for a prospective component-resolved diagnosis of Type I allergy, and the basis for the design and monitoring of a patient-tailored specific immunotherapy in the future.     

Screening the allergenic repertoires of wheat and maize with sera from double-blind, placebo-controlled food challenge positive patients

BACKGROUND: Food allergy to wheat and maize is an increasing factor of deterioration of life quality, especially childhood and can, in rare cases, even induce anaphylaxis. Although omega-5 gliadin from wheat and maize lipid transfer protein have been characterized as major cereal allergens on the molecular level, the list of food allergens is far to be complete. METHODS: To identify the IgE-binding repertoires of wheat and maize we screened respective cDNA libraries displayed on phage surface with sera from patients with a confirmed food allergy. The study included six patients with a positive double-blind, placebo-controlled food challenge (DBPCFC) to wheat, nine patients with a positive DBPCFC to maize, and six patients with anaphylactic reactions after ingestion of wheat. RESULTS: The enriched sequences encoding IgE-binding proteins showed heterogeneous repertoires for both, wheat and maize. The selected wheat repertoire yielded 12, the maize repertoire 11 open reading frames. Among these we identified allergens belonging to already characterized allergens families, such as gliadin, profilin and beta-expansin. Besides, we found novel proteins with high cross-reactive potential, such as thioredoxins, as well as sequences that had so far not been related to cereal allergy at all. The IgE-binding capacity of some selected proteins was evaluated in vitro and cross-reactivity was demonstrated by competition ELISA. CONCLUSION: With regard to the heterogeneity of the characterized sequences as well as to the biochemical nature of the new allergens detected we conclude that wheat and maize-related food allergy is more complex than so far anticipated.     

Diagnosis of Alternaria alternata sensitization with natural and recombinant Alt a 1 allergens

BACKGROUND: Diagnosis of Alternaria alternata sensitization is hampered by the variability and complexity of fungal extracts, and thus simplification of the diagnostic procedures with purified allergens should be pursued. OBJECTIVE: We sought to compare A alternata extract and purified natural Alt a 1 (nAlt a 1) and recombinant Alt a 1 (rAlt a 1) allergens for their diagnostic value. METHODS: Forty-two patients allergic to A alternata , 10 atopic patients with negative skin prick test responses to A alternata extract, and 10 healthy subjects were investigated. Skin prick tests and determination of specific IgE levels were performed with nAlt a 1 and 2 different types of rAlt a 1: rbAlt a 1, expressed in Escherichia coli , and ryAlt a 1, expressed in the yeast Yarrowia lipolytica . RESULTS: Prevalence for Alt a 1, Alt a 2, and Alt a 11 by IgE dot-blot testing was 98%, 0%, and 15%, respectively, and therefore Alt a 1 was used as a marker for A alternata sensitization. Immunoblotting and inhibition analysis showed no IgE-binding differences between nAlt a 1 and rAlt a 1. The whole group of patients with allergy to A alternata had positive skin test reactions to purified allergens at 100 microg/mL, whereas no false-positive reactions were detected. Natural or ryAlt a 1 elicited a similar response in skin tests compared with A alternata extract, although a reduced reactivity was observed with rbAlt a 1. Specific IgE levels to nAlt a 1 or rAlt a 1 showed significant correlation and similar sensitivity and specificity. CONCLUSIONS: Alt a 1, either in its natural or recombinant form, is sufficient for a reliable diagnosis of A alternata sensitization and induces skin prick reactivity comparable with that produced by A alternata extract.     

人二型大麻受体激动剂高通量筛选模型的建立

目的:基于二型大麻受体(CB2)的信号传递通路,建立体外高通量筛选CB2受体激动剂的药物模型。方法:将目的基因质粒pIRES2-EGFP-CB2、报告基因质粒pGL4.29[luc2P/CRE/Hygro]、内参质粒PRL-TK共转染CHO细胞,通过检测双荧光素酶报告基因的表达水平变化来筛选人CB2受体的激动剂。并对加入激动剂的浓度和作用时间进行优化及考察模型的稳定性。结果:给予10μmol/L JWH-015 6 h后能取得最大相对诱导率。成功建立CB2受体激动剂的高通量筛选模型,筛选模型Z’因子为0.81,表现为良好的稳定性。结论:成功地建立了CB2受体激动剂的筛选模型,为从中药中高通量筛选有效物质奠定了良好的基础。     

Reactivity to recombinant house-dust-mite allergens in asthma and rhinitis in a tropical environment

Background House-dust mites contain components that are allergenic in mite-sensitive patients, and a number of these have been produced in recombinant form.
Methods In the present study, we evaluated by skin prick testing the positivity to native Der p 2 and recombinant Der p 2, Der p 5, and Der p 7 allergens of Dermatophagoides pteronyssinus in patients with rhinitis, asthma, or a combination of these diseases, who were positive to whole-mite extract. Results In all patients, the positivity to both native and recombinant Der 2 was high. In patients with either rhinitis or asthma, the reactivity to Der p 5 and 7 was significantly lower than to Der p 2. However, in patients with combined disease, the positivity to the minor allergens was almost as high as that to Der p 2.
Conclusions These results raise the question of whether patients with combined allergic rhinitis and asthma, when compared to those with either of these diseases alone, are predisposed to react to a wider range of mite allergens, or, inversely, whether patients who respond to the minor allergens are more susceptible to suffering the combined disease.     

Fagales pollen sensitization in a birch-free area: a respiratory cohort survey using Fagales pollen extracts and birch recombinant allergens (rBet v 1, rBet v 2, rBet v 4)

BACKGROUND: Birch allergy is one of the most common pollinosis in areas where exposure to high levels of birch pollen is common. Little is known about birch sensitivity in areas without birch pollen exposure and reactivity to birch-related species within the Fagales order. OBJECTIVE: the aim was to evaluate Fagales reactivity within a population not exposed to birch pollen using epidemiological, diagnostic, and laboratory approaches by means of extracts and allergenic molecules. METHODS: A cohort of 5335 respiratory allergic patients was screened by means of skin testing birch, hazel, and oak pollen extracts. Patients were from a birch-free area, but exposed to other Fagales pollen species. A subset of patients was from an intensively cultivated hazel area. A sample of the Fagales allergic population was tested with other Fagales pollen extract (alder, hornbeam, beech, chestnut) and with apple and hazelnut. IgE detection was performed with birch, hazel, oak, apple, and hazelnut extracts, and with Bet v 1, Bet v 2, Bet v 4, and bromelain. IgE immunoblots were performed using birch and hazel extracts. Epidemiological, clinical, and laboratory data were analysed by stratifying the allergic population. RESULTS: Twenty-five percent of the pollen allergic cohort was skin test positive to at least one of the three Fagales species. Combined reactivity to the three species was recorded in 80% of this cohort. Isolated hazel pollen reactivity was recorded in 13.5% of the Fagales allergic patients. Sixty-six percent of these subjects were from the intensively cultivated hazel area. Reactivity to apple and hazelnut was detected by skin test (40%) and IgE reactivity (60%), but only 19% of the positive patients reported symptoms related to at least one of the two foods. Reactivity to Bet v 1 was recorded in 84% of the birch/hazel/oak co-reactivity group, and in 28% of the subjects with the same co-reactivity, but associating a multiple pollen sensitization. IgE to Bet v 2 (50%) and Bet v 4 (23%) panallergens were recorded positive in the latter subset. Bet v 1 prevalence ranged between 48% and 21% among subgroups of patients coming from different areas. Furthermore, an IgE reactivity to hazel-restricted allergenic components was detected among subjects coming from the same area and having a hazel isolated reactivity. CONCLUSION: Fagales allergy can be found in birch-free areas caused by the exposure to other Fagales species. Birch allergens can be useful for mimicking the allergenic extract, but are also the exclusive tools for a fine diagnostic and epidemiological approach to Fagales pollen allergy. Allergenic molecules from the hazel family will increase the panel of available reagents for the molecule-based approach to allergy diagnosis and therapy.     

Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy

BACKGROUND: The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts. OBJECTIVE: To produce recombinant dog allergens Can f 1 and Can f 2 in Pichia pastoris yeast and to assess their suitability for the diagnosis of dog allergy. METHODS: Clinically diagnosed dog-allergic patients' and healthy non-atopic dog owners' reactivities against recombinant Can f 1 and Can f 2 and commercial dog epithelial extract were studied by a panel of methods including skin prick test (SPT), ELISA and IgE immunoblotting. RESULTS: Recombinant Can f 1 and Can f 2 were found immunologically functional: they bound dog-allergic patients' IgE in immunoblotting and inhibited specifically the binding of IgE to their natural counterparts in the dog allergen extract. Moreover, patients' IgE reactivity in immunoblotting to natural Can f 1 and their SPT with the recombinant allergen were perfectly concordant (phi coefficient 1.0, P<0.001). The concordance was slightly lower with recombinant Can f 2 (phi coefficient 0.92, P<0.001). A lower number of dog-allergic patients, 52%, reacted against Can f 1 than previously reported. About one-third of the patients reacted to Can f 2. In immunoblotting, the highest prevalence of reactivity, 60%, was directed to an 18 kDa component. Aminoterminal sequencing showed this to be a previously unidentified allergenic protein. CONCLUSIONS: The recombinant allergens can be used reliably to identify Can f 1 and Can f 2-sensitized individuals. However, the two allergens are insufficient as reagents for diagnosing dog allergy.     

Monitoring allergen immunotherapy of pollen-allergic patients: the ratio of allergen-specific IgG4 to IgG1 correlates with clinical outcome

BACKGROUND: Although allergen immunotherapy has been established as a treatment of type I allergy back in 1911, until now the underlying mechanisms have not been fully understood, nor are there any parameters which would allow one to monitor an ongoing treatment or to assess therapeutic success in the meantime. OBJECTIVE: We wanted to define allergen-specific parameters that change due to treatment in correlation with the clinical outcome. METHODS: We conducted a controlled study with grass pollen-allergic children and compared allergen-specific antibody titres before and 1 year after the onset of immunotherapy in contrast with untreated allergic and healthy children. Two recombinant forms of the major allergen group V of Phleum pratense (Phl p 5) served as model allergens. RESULTS: No change in IgE levels and no significant reduction of skin prick test (SPT) reactivity were seen. On the other hand, a significant reduction of symptom scores in the treated group and a significant rise in allergen-specific IgG1, IgG2 and IgG4 due to the treatment could be observed, but in neither case could we establish a correlation between the increasing amounts of the single antibody classes and the reduction of symptom scores. But most interestingly, when comparing the ratio of IgG4 to IgG1 with the symptom scores, we found significant correlations. Nevertheless, treated allergic patients still differ considerably from healthy controls as nonatopics have hardly any measurable allergen-specific IgG antibodies and no IgE antibodies at all. CONCLUSION: The ratio of IgG4 to IgG1 can serve as a valuable parameter that allows us to assess the success of immunotherapy already 1 year after the onset. The increase of specific IgG1 in relation to IgG4 during treatment reflects a possible influence of this subclass on the induction of tolerance towards allergens.     

Integrating high‐throughput screening and sequencing for monoclonal antibody discovery and engineering

Monoclonal antibody discovery and engineering is a field that has traditionally been dominated by high‐throughput screening platforms (e.g. hybridomas and surface display). In recent years the emergence of high‐throughput sequencing has made it possible to obtain large‐scale information on antibody repertoire diversity. Additionally, it has now become more routine to perform high‐throughput sequencing on antibody repertoires to also directly discover antibodies. In this review, we provide an overview of the progress in this field to date and show how high‐throughput screening and sequencing are converging to deliver powerful new workflows for monoclonal antibody discovery and engineering.     

Selected recombinant Aspergillus fumigatus allergens bind specifically to IgE in ABPA

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease resulting from exposure to Aspergillus fumigatus allergens. Patients with ABPA show elevated Aspergillus-specific serum IgE, a major criterion used in the diagnosis of the disease. Crude culture filtrate and mycelial antigens have been used widely to demonstrate IgE antibody to Aspergillus in the sera of patients. While these antigens have been useful in the diagnosis of ABPA, occasionally they present inconsistency in their reactivity and lack of specificity. Although in recent years, a number of purified A. fumigatus allergens have been produced by molecular cloning, no attempt was made to evaluate them systematically. OBJECTIVE: To evaluate the recombinant proteins from A. fumigatus for their IgE antibody binding, we studied sera from ABPA patients and controls by antigen specific enzyme linked immunosorbent assay (ELISA). METHODS: Recombinant Aspergillus allergens Asp f 1, f 2, f 3, f 4, and f 6 were studied for their specific binding to IgE in the sera of ABPA patients, A. fumigatus skin prick test positive asthmatics, and normal controls from the USA and Switzerland. The sera were blinded and studied by ELISA in two different laboratories. RESULTS: All the recombinant allergens showed IgE antibody binding with sera from patients with ABPA, whereas only fewer asthmatics and normal sera showed significant binding. The three selected recombinant allergens together reacted with all the ABPA patients studied. CONCLUSIONS: The results demonstrate that Asp f 2, f 4, and f 6 can be used in the serodiagnosis of ABPA, while IgE antibody binding to Asp f 1 and f 3 was not specific.     

IgE antibody to Aspergillus fumigatus recombinant allergens in cystic fibrosis patients with allergic bronchopulmonary aspergillosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) is characterized by a heightened Th2 CD4+ T-cell response to Aspergillus fumigatus (Af) allergens and a hyper-immunoglobulin E (IgE) state compared with cystic fibrosis patients without ABPA. The IgE serologic differentiation of ABPA from atopic CF patients can be difficult. We propose as the reactivity with purified antigens varies qualitatively and quantitatively and that the antibody response is more specific than with crude Af antigen extract, the IgE responses to purified recombinant Af allergens may differentiate ABPA from atopic CF patients. METHODS: Serum IgE reactivity to seven recombinant purified allergens and to a crude extract of Af was measured in 15 ABPA, in 23 Af skin test positive (ST+), and in 19 Af skin test negative (ST-) CF patients. Four of the ABPA CF patients were studied before and after developing ABPA. Nine ABPA patients were studied during flares and remissions of ABPA. RESULTS: Allergic bronchopulmonary aspergillosis patients had significantly increased IgE reactivity to Asp f2, f3, f4, f6, and f16 compared with the Af ST+ and ST- non-ABPA CF patients. In the ABPA patients studied before and after developing ABPA, IgE reactivity also increased to Asp f2, f3, f4, and f6, and to the crude extract. In ABPA CF patients, IgE reactivity to Asp f1, f2, f3, and f6 significantly increased during periods of ABPA flares compared with periods of remission. Analysis of the receiver operating curve demonstrated that IgE reactivity to Asp f3 and f4 gave the best sensitivity and specificity and were better than IgE reactivity to a crude extract of Aspergillus. Furthermore, in ABPA patients studied during periods of remission the IgE reactivity to Asp f3 and f4 remained significantly elevated compared with Af ST+ non-ABPA patients. The IgE responses when considered either to be positive or negative to Asp f3 and f4 significantly differentiated ABPA from Af ST+ and ST- non-ABPA CF patients. In contrast, IgE reactivity was considered positive to the crude extract in 89% of ABPA, 61% of Af ST+, and 0% of Af ST- non-ABPA CF patients. CONCLUSIONS: Immunoglobulin E reactivity to a panel of purified Af allergens, especially to Asp f3 and f4, differentiates ABPA from atopic Af ST+ non-ABPA CF patients. Serial determinations of IgE reactivity to individual purified Aspergillus antigens, especially Asp f3, demonstrates that increases in IgE reactivity may provide improved distinction between stages of flares and remission compared with changes in IgE reactivity to a crude Aspergillus extract.