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Cloning Aspergillus fumigatus Allergens by the pJuFo Filamentous Phage Display System
Abstract
A cDNA library from Aspergillus fumigatus has been displayed on the surface of filamentous phage M13 and screened for gene products binding to human serum IgE. The physical linkage of cDNA gene products to the genetic information required for their production, achieved by exploiting the high-affinity interaction of the Jun and Fos leucine zippers, allows rapid and easier screening of large libraries in semifluid systems. The pJuFo cloning vector is designed to display proteins on the surface of phage and allows selective isolation of genes by gene product-ligand interaction. Thus the system is applicable to clone cDNA that encodes proteins for which a ligand is available. Herein we show that phage expressing IgE binding proteins from A. fumigatus can be enriched and separated from nonspecific phage by using serum IgE from A. fumigatus-allergic individuals coated to plastic dishes. Subsequently, the proteins can be produced in high amounts in Escherichia coli and purified for usage in allergy testing.
Supplementary resource (1)
... These observations indicate that a specific sensitization to single A. fumigatus allergens can occur and could potentially be exploited to improve the diagnosis of ABPA (26) and/or to characterize the stages of the disease (27). We have cloned, characterized and produced a panel of A. fumigatus allergens (28) using a new cloning system based on phage surface display technology (28)(29)(30) to study the differential IgE antibody responses of A. fumigatus-sensitized asthmatics to single allergens and to evaluate the possibility of using recombinant allergens to confirm a clinical suspicion of ABPA. ...
... The ribotoxin rAsp f 1 (22,23), rAsp f 3, a protein homologous to peroxisomal proteins of Candida boidinii (33), rAsp f 4, an allergen with unknown biological function, and manganese dependent superoxide dismutase (rAsp f 6) (34) were cloned from an A. fumigatus cDNA library displayed on phage surface (28). The regions coding for the mature proteins (22,28,33,34) were subcloned into the p[His] 6 -DHFR high level expression vector, constructs verified by nucleotide sequence determinations and used to produce hexahistidinetagged recombinant proteins in Escherichia coli as described (22,34). ...
... The ribotoxin rAsp f 1 (22,23), rAsp f 3, a protein homologous to peroxisomal proteins of Candida boidinii (33), rAsp f 4, an allergen with unknown biological function, and manganese dependent superoxide dismutase (rAsp f 6) (34) were cloned from an A. fumigatus cDNA library displayed on phage surface (28). The regions coding for the mature proteins (22,28,33,34) were subcloned into the p[His] 6 -DHFR high level expression vector, constructs verified by nucleotide sequence determinations and used to produce hexahistidinetagged recombinant proteins in Escherichia coli as described (22,34). The fusion proteins were purified in a single step by Ni 2ϩ -chelate affinity (22,33). ...
Allergic bronchopulmonary aspergillosis (ABPA), a severe pulmonary complication caused by Aspergillus fumigatus, is considered a complex clinical syndrome with defined serological, pathological radiological and clinical features. The diagnosis of ABPA is often difficult because of several overlapping clinical and laboratory findings shared between asthma with sensitization to A. fumigatus and ABPA, but essential for treatment to prevent severe deterioration of pulmonary function. We have cloned A. fumigatus allergens from a cDNA library displayed on phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. The single recombinant allergens were used to assess the immunological response in representative groups of A. fumigatus-sensitized asthmatics with or without ABPA and healthy controls. The allergens rAsp f 1, a 16.9 kDa ribotoxin, rAsp f 3, a 18.5 kDa peroxisomal protein, and rAsp f 6, a 23 kDa manganese superoxide dismutase, were identified as proteins with known biological function and rAsp f 4, a 30 kDa allergen, lacks sequence homology to known proteins. The secreted ribotoxin rAsp f 1 and rAsp f 3 are recognized by serum IgE of A. fumigatus-sensitized asthmatics with or without ABPA, whereas the non-secreted manganese superoxide dismutase rAsp f 6 and the rAsp f 4 allergen are exclusively recognized by serum IgE of ABPA patients. The dissection of IgE-mediated immune responses to single recombinant A. fumigatus allergens in asthmatic patients allow a discrimination between ABPA and A. fumigatus sensitization with high specificity (100%) and sensitivity (90%).
... Phage displaying IgE-binding proteins were enriched from an A. fumigatus cDNA library constructed in phagemid pJuFo (19) and displayed on the surface of filamentous phage M13 as described (5, 6). Serum IgE from A. fumigatus–sensitized individuals was captured in microtiter plates coated with anti– human mAb TN 142 and used as ligand for selective enrichment of allergen-displaying phage (6). Sera used for screening were selected according to case history, skin test reactivity to commercial A. fumigatus extracts, and specific IgE to A. fumigatus determined by radioallergosorbent test (RAST) (8). ...
... Ligation mixtures were transformed into Escherichia coli strain M 15; transformants were grown in liquid to verify the nucleotide sequence (23) and used to produce hexahistidine-tagged recombinant proteins (4, 6). After a single-step purification over Ni2+–chelate affinity columns (6), molecular size and purity of the recombinant proteins were analyzed by polyacrylamide gradient gels (4.5–20%) and 1-mg samples lyophilized for long term storage (14). ...
... Sera used for screening were selected according to case history, skin test reactivity to commercial A. fumigatus extracts, and specific IgE to A. fumigatus determined by radioallergosorbent test (RAST) (8). The screening procedure yielded a wide variety of phage able to bind specifically to human serum IgE and thus displaying allergenic molecules (5, 6). ...
A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus cDNA library displayed on the surface of filamentous phage. Solid phase–immobilized serum immunoglobulin E (IgE) from A. fumigatus–allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein
that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed rAsp f 8, shares >62% sequence identity and >84% sequence homology to corresponding
eukaryotic P2 proteins, including human P2 protein. The sequences encoding human and fungal P2 protein were subcloned, expressed in Escherichia coli as His6-tagged fusion proteins, and purified by Ni2+–chelate affinity chromatography. Both recombinant P2 proteins were recognized by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited strong type 1–specific skin reactions in these individuals. Moreover, human and fungal P2 proteins induced proliferative responses in peripheral blood mononuclear cells of A. fumigatus– allergic subjects sensitized to the fungal P2 protein. These data provide strong evidence for in vitro and in vivo humoral and cell-mediated autoreactivity to human P2 protein in patients suffering from chronic A. fumigatus allergy.
... Among these species, A. fumigatus is implicated in approximately 80% of Aspergillus-related infections (Simon-Nobbe et al. 2008). Due to its high prevalence, a large number of allergens from A. fumigatus have been cloned, characterised and purifi ed as recombinant allergens (Crameri and Blaser 1996;Crameri et al. 2006). ...
... Other allergens from Aspergillus have been described, including Asp f 2, Asp f 4 and Asp f 6 (Kurup and Banerjee 2000; Vijay and Kurup 2004), and are available as recombinant proteins (Crameri and Blaser 1996). Asp f 4 and Asp f 6 are intracellular proteins; thus, the availability of these proteins as aeroallergens is unlikely. ...
... Results showed that the gene-disrupted mutants for Asp f5 of A. fumigatus retained virulence and it has been considered to be involved in degradation of lung structural architecture by enzymatic activity [89]. Crameri et al. (1996) used serum IgE antibodies from A. fumigatus-sensitized individuals to enrich phageexpressing allergenic proteins [90]. They identified one cDNA encoding for 26.7 kDa manganese superoxide dismutase called Asp f6 sharing 67.2% homology with human manganese superoxide dismutase which also showed autoreactivity in allergic persons sensitized to Asp f6 of environmental A. fumigatus. ...
... Results showed that the gene-disrupted mutants for Asp f5 of A. fumigatus retained virulence and it has been considered to be involved in degradation of lung structural architecture by enzymatic activity [89]. Crameri et al. (1996) used serum IgE antibodies from A. fumigatus-sensitized individuals to enrich phageexpressing allergenic proteins [90]. They identified one cDNA encoding for 26.7 kDa manganese superoxide dismutase called Asp f6 sharing 67.2% homology with human manganese superoxide dismutase which also showed autoreactivity in allergic persons sensitized to Asp f6 of environmental A. fumigatus. ...
Incidence of fungal infections has increased alarmingly in past few decades. Of the fungal pathogens, the Aspergillus fumigatus has been a major cause of allergic bronchopulmonary aspergillosis (ABPA) which has five main stages - the acute, remission, exacerbation, glucocorticoid dependent and fibrotic stage. The diagnosis of ABPA remains difficult due to its overlapping clinical and radiological features with tuberculosis and cystic fibrosis. From past few decades, the crude fractions of A. fumigatus have been used for immunodiagnosis of ABPA. Most of the detection kits based on crude fractions of A. fumigatus are quite sensitive but have low specificity. Till date 21 known and 25 predicted allergens of A. fumigatus have been identified. Of these allergens, only five recombinants (rAsp f1-f4 and f6) are commercially used for diagnosis of allergic aspergillosis. Remaining allergens of A. fumigatus have been restricted for use in specific diagnosis of ABPA, due to sharing of common antigenic epitopes with other allergens. Complete sequencing of A. fumigatus genome identified 9926 genes and the reports on the proteome of A. fumigatus have shown the presence of large number of their corresponding proteins in the pathogen. The analysis of immunoproteomes developed from crude fractions of A. fumigatus by IgG/IgE reactivity with ABPA patients and animal sera have identified the panel of new antigens. A brief description on the current status of A. fumigatus antigens is provided in this review. The implementation of advance recombinant expression and peptidomic approaches on the A. fumigatus antigens may help in the selection of appropriate molecules for the development of tools for more specific early diagnosis of ABPA, and desensitization therapies for patients of allergic disorders.
... Recombinant allergens are definitively more reliable for diagnostic applications as shown for rAsp f 1, a major allergen from A. fumigatus, in studies involving patients suffering from asthma or CF [10][11][12]. We have cloned, characterized, and produced a panel of allergens from A. fumigatus [13,14] and demonstrate that CF patients with ABPA mount specific IgE responses against two allergens of the fungus which are not recognized by A. fumigatus-sensitized CF patients without ABPA. ...
... The four cDNA encoding A. fumigatus allergens used to produce hexahistidine-tagged N-terminal fusion proteins in Escherichia coli yielded between 36 and 220 mg/l culture depending on the cDNA insert [14]. The affinitypurified recombinant allergens were virtually pure as shown by SDS gel electrophoresis and silver staining (not shown) and free of IgE-binding contaminants as shown by Western blot analysis (Fig. 1). ...
Allergic bronchopulmonary aspergillosis (ABPA), an intense inflammatory reaction to Aspergillus in the lung, is recognized as a severe complication in patients with cystic fibrosis (CF). The diagnosis of ABPA in CF patients sensitized to Aspergillus fumigatus is complicated by interfering laboratory and clinical findings shared by the diseases. We have used cDNA encoding A. fumigatus allergens which were cloned from a cDNA library displayed on phage surface to produce recombinant proteins in Escherichia coli. Differential IgE responses to the allergens in A. fumigatus-sensitized CF patients with or without ABPA and CF controls without sensitization to A. fumigatus were demonstrated. A secreted ribotoxin (rAsp f 1) and a peroxisomal protein (rAsp f 3) were recognized by sera from A. fumigatus-sensitized CF-patients with or without ABPA. An intracellular manganese superoxide dismutase (rAsp f 6) and rAsp f 4, a protein with unknown function, were recognized exclusively by IgE from sera of CF patients with ABPA. Therefore, Asp f 4 and Asp f 6 represent specific markers for ABPA and allow a sensitive, fully specific diagnosis of the disease. The data suggest distinct IgE responses to colonization of the bronchial tree in CF patients with ABPA or A. fumigatus allergy and therefore a differential recognition of the pathogen in the two IgE-related inflammatory diseases.
... However, only three out of six well-characterized ABPA patients had positive skin prick test reactions to r Asp f 1, although all patients reacted to crude A. fumigatus preparations. Further recombinant A. fumigatus allergens have been cloned, expressed, and purified (24,25). The most important among these are r Asp f 3, a 18.5-kD peroxisomal protein, r Asp f 4, a 30-kD protein of unknown biologic function, and r Asp f 6, a 23-kD manganese superoxide dismutase. ...
... The cDNA gene products were covalently linked to the surface of the phage by the strong interaction of the Jun and Fos leucine zippers, allowing a rapid and simplified screening of cDNA libraries by gene product-ligand interaction. Phages expressing IgE-binding proteins from A. fumigatus were selectively enriched from unspecific phages using serum IgE from A. fumigatus -allergic individuals, immobilized to solid phase supports (24,25). Subsequently, the proteins were produced in E. coli and purified for usage in allergy testing (32). ...
Before specific therapy, such as oral corticosteroids, can be commenced it is essential to distinguish full-blown allergic bronchopulmonary aspergillosis (ABPA) from allergy to A. fumigatus in patients with cystic fibrosis (CF). For this purpose we have evaluated the diagnostic value of recombinant A. fumigatus allergen I/a (rAsp f I/a)-specific serology in 55 patients with CF. Based on clinical presentation and laboratory data, 10 CF patients had ABPA, 27 had Aspergillus allergy, and 18 were not allergic to A. fumigatus (CF control group). The serologic assays revealed a 10-fold increase in rAsp fI/a-specific IgE, a 5-fold increase in rAsp fI/a-specific IgG1, and a 4-fold increase in rAsp fI/a-specific IgG4 antibodies in ABPA patients compared with the Aspergillus allergy and CF control groups. Sera from 11 CF patients were analyzed without knowledge of their clinical state or diagnosis of ABPA. All ABPA patients (4 of 11) were accurately identified. We conclude that rAsp fI/a-specific serology is a highly sensitive and specific test that can be used to identify ABPA reliably in CF patients.
... Recombinant allergens are definitively more reliable for diagnostic applications as shown for rAsp f 1, a major allergen from A. fumigatus, in studies involving patients suffering from asthma or CF [10][11][12]. We have cloned, characterized, and produced a panel of allergens from A. fumigatus [13,14] and demonstrate that CF patients with ABPA mount specific IgE responses against two allergens of the fungus which are not recognized by A. fumigatus-sensitized CF patients without ABPA. ...
... The four cDNA encoding A. fumigatus allergens used to produce hexahistidine-tagged N-terminal fusion proteins in Escherichia coli yielded between 36 and 220 mg/l culture depending on the cDNA insert [14]. The affinitypurified recombinant allergens were virtually pure as shown by SDS gel electrophoresis and silver staining (not shown) and free of IgE-binding contaminants as shown by Western blot analysis (Fig. 1). ...
Allergic bronchopulmonary aspergillosis (ABPA), an intense inflammatory reaction to Aspergillus in the lung, is recognized as a severe complication in patients with cystic fibrosis (CF). The diagnosis of ABPA in CF patients sensitized to Aspergillus fumigatus is complicated by interfering laboratory and clinical findings shared by the diseases. We have used cDNA encoding A. fumigatus allergens which were cloned from a cDNA library displayed on phage surface to produce recombinant proteins in Escherichia coli. Differential IgE responses to the allergens in A. fumigatus-sensitized CF patients with or without ABPA and CF controls without sensitization to A. fumigatus were demonstrated. A secreted ribotoxin (rAsp f 1) and a peroxisomal protein (rAsp f 3) were recognized by sera from A. fumigatus-sensitized CF-patients with or without ABPA. An intracellular manganese superoxide dismutase (rAsp f 6) and rAsp f 4, a protein with unknown function, were recognized exclusively by IgE from sera of CF patients with ABPA. Therefore, Asp f 4 and Asp f 6 represent specific markers for ABPA and allow a sensitive, fully specific diagnosis of the disease. The data suggest distinct IgE responses to colonization of the bronchial tree in CF patients with ABPA or A. fumigatus allergy and therefore a differential recognition of the pathogen in the two IgE-related inflammatory diseases.
... The method used for the construction of the pJuFo surface display library was as described. 13 λ-ZapII cDNA library 100 times the primary size of C comatus were excised in vivo. A total of 4.5 µg cDNA inserts, obtained by Xba1/Kpn1 digestion of the excised phagemid, were gel purified, electroeluted, and ligated to 1.5 µg of Xba1/Kpn1-restricted pJuFo plasmid. ...
... Just before the screening process this library was freshly amplified 13 and subjected to 5 rounds of panning against serum IgE from C comatus-sensitized individuals as ligand, immobilized on solid phase with antihuman IgE mAb TN-142 as the capturing antibody. 13 The serum used for screening was obtained by pooling serum of 4 C comatus-sensitized subjects. 8 ...
Basidiomycetes spores are ubiquitously distributed, found throughout the year in outdoor and indoor air, and represent relevant sources of aeroallergens associated with allergy and asthma.
Cloning and characterization of Coprinus comatus (shaggy cap mushroom) allergens is essential to elucidate their molecular characteristics and to improve the diagnosis of allergy.
A complementary DNA (cDNA) library of C comatus displayed on phage surface was screened with sera of basidiomycete-sensitized individuals. Subcloning and high-level expression of one of the enriched cDNAs allowed the isolation of a [His](6)-tagged recombinant protein formally termed rCop c 1. The allergenic properties of rCop c 1 were investigated in vitro by ELISA, inhibition experiments, immunoblots, and proliferation assays and in vivo by skin tests.
The rCop c 1-encoding cDNA spans 435 bp and contains an open reading frame of 246 bp, predicting a protein of 8.96 kd without significant sequence homology to known proteins. Immunoblots with [His](6)-rCop c 1 fusion protein show a background free IgE-binding band of the expected size that can be completely inhibited by crude C comatus extracts in ELISA. rCop c 1 induced specific proliferative responses in PBMCs of C comatus-sensitized individuals. The incidence of sensitization to rCop c 1 among 92 sera of basidiomycete-sensitized individuals tested in ELISA was 25%, indicating that Cop c 1 is an intermediate allergen. However, prick tests showed that less than 2 pmol of the rCop c 1 protein was able to induce strong specific skin reactions in sensitized individuals.
rCop c 1, the first cloned allergen from the genus Coprinus, fulfills all the criteria required to be classified as a clinically relevant allergen. The data demonstrate at the molecular level the presence of sensitizing molecules among Basidiomycetes, the most important source contributing to the total spore load in the outdoor air.
... 12 Using a special phage surface display technology, 13 we have cloned a panel of recombinant A fumigatus allergens. 12,14 Thus far it has been shown that patients with ABPA, compared with A fumigatus-sensitized asthmatic subjects, exhibit significantly elevated specific IgE values against Asp f 1 and Asp f 3, 2 major allergens of A fumigatus. 15,16 In both cases the IgE levels in A fumigatus-sensitized patients with or without ABPA to these 2 allergens were strongly overlapping and do not allow a reliable discrimination between these 2 diseases. ...
... We used a phage surface display-based cloning system, which allows rapid cloning, sequencing, and production of IgE-binding molecules, [12][13][14] to clone a panel of A fumigatus allergens. 12 The major allergens rAsp f 1 and rAsp f 3 were investigated in large serologic and clinical studies. ...
Aspergillus fumigatus, an opportunistic pathogen, is associated with an impressive list of pulmonary complications. Among these, allergic bronchopulmonary aspergillosis (ABPA) represents a complex clinical syndrome that is difficult to diagnose. A clear distinction between allergic sensitization to A fumigatus and ABPA is essential for therapy to prevent deterioration of pulmonary function in subjects with ABPA.
This study was carried out to determine the specificity and sensitivity of 2 A fumigatus allergens for the in vivo diagnosis of ABPA.
Serologic investigations with recombinant A fumigatus allergens indicated the existence of disease-specific allergens that are useful for discrimination between ABPA and fungal sensitization. However, serologic studies fail to indicate the allergen-specific IgE levels required to elicit an allergic reaction in vivo.
We show that the recombinant A fumigatus allergens rAsp f 4, a protein with unknown biologic function, and rAsp f 6 (manganese superoxide dismutase) are able to provoke immediate skin reactions exclusively in patients with ABPA. The reactions, which are elicited by a few nonograms of the allergens, strictly depend on the presence of allergen-specific serum IgE. The IgE cut-off values for positive skin reactions to rAsp f 4 and rAsp f 6 of 0.9 and 1.2 kU(A)/L correspond to allergen-specific serum concentrations of 2 to 3 microg/L and allow a sensitive, highly specific diagnosis of ABPA.
In contrast to fungal extracts, rAsp f 4 and rAsp f 6 allow discrimination between ABPA and sensitization to A fumigatus. Moreover, the allergens are suitable for an automated serologic diagnosis of ABPA, facilitating their introduction in clinical practice.
... Another previously found Aspergillus fumigatus allergen, Asp F 6, is a manganese-dependent superoxide dismutase (MnSOD), which catalyzes the 204 conversion of superoxide radicals to hydrogen peroxide and oxygen.[174][175] Due to the specific presence of Asp F 6 in ABPA patient, it became a potential precise diagnosis of ABPA[176][177][178] , a condition with an exaggerated response of the immune system to the fungus Aspergillus (most commonly Aspergillus fumigatus). Several studies[179][180][181] were conducted to identify antigenic proteins through immunoblotting of Aspergillus fumigatus mycelial extract with sera from patients with IA, or rabbit infected with Aspergillus fumigatus. ...
... Another previously found Aspergillus fumigatus allergen, Asp F 6, is a manganese-dependent superoxide dismutase (MnSOD), which catalyzes the 204 conversion of superoxide radicals to hydrogen peroxide and oxygen.[174][175] Due to the specific presence of Asp F 6 in ABPA patient, it became a potential precise diagnosis of ABPA[176][177][178] , a condition with an exaggerated response of the immune system to the fungus Aspergillus (most commonly Aspergillus fumigatus). Several studies[179][180][181] were conducted to identify antigenic proteins through immunoblotting of Aspergillus fumigatus mycelial extract with sera from patients with IA, or rabbit infected with Aspergillus fumigatus. ...
Mass spectrometry (MS) has become a powerful tool in biological sciences research. This dissertation reports the progress made in uncovering the effect of Salmonella on the intestinal environment by MS-based quantification and characterization of small molecules, and in characterizing a Salmonella deglycase that is a potential drug target. The MS- inspired discovery of protein biomarkers in Aspergillus fumigatus that causes invasive aspergillosis (IA) is also documented. Chapters 2, 3, and 4 focus on Salmonella enterica serovar Typhimurium, one of the major causes of food-borne diseases. The hypothesis of Chapter 2 is that the use of metabolomics, metagenomics, and metatranscriptomics will uncover the effect of Salmonella enterica serovar Typhimurium on the gastrointestinal environment, specifically in terms of biomolecules, microbiota, and host cells and tissues. Untargeted metabolite analysis using liquid chromatography-mass spectrometry (LC-MS), shows that the Salmonella-infected intestine has a significantly different distribution of nutrients and metabolites compared with that for uninfected animals, which is consistent with the shift in the distribution of microbes. The observed decrease of short-chain fatty acids (SCFA) was also validated by using targeted analysis, and is consistent with the decrease in Clostridia, a well-known SCFA producer. This multi-omics approach can be applied to discovering prebiotics and probiotics to prevent Salmonella infection. An intermediate of the fructose-asparagine (F-Asn) pathway, unique in Salmonella and few other species, has been found to be toxic to a fraB mutant of Salmonella. Results shown in Chapters 3 and 4 lay the foundation for a new strategy to specifically inhibit Salmonella using the combination of a FraB inhibitor and F-Asn. Chapter 3 describes the quantification of F-Asn in various foods, using a hydrophilic interaction liquid chromatography (LC) coupled to a triple-quadrupole mass spectrometer. F-Asn is present in a wide range of human and animal foods, and is transported to the gastrointestinal tract, where it can be consumed by Salmonella and a few other microorganisms. These results pave the way for the study of dosing of F-Asn in a drug cocktail. Chapter 4 focuses on studying the native structure and catalytic mechanism of FraB. Native MS analysis was performed on purified recombinant FraB. The oligomeric state, collision-cross section, dimer interface, and ligand-binding stoichiometry derived from MS data support a homology model proposed by our collaborators. MS also supports that various site-directed FraB mutants preserve the native structure, suggesting that the loss of enzymatic activity in these mutants does not result from global structural alterations. The successful detection of the binding between FraB and its substrate or products can be transferred to study FraB-inhibitor interaction. The discovery of protein biomarkers for Aspergillus fumigatus infection in IA diagnosis is discussed in Chapter 5. A reversed-phase/reversed-phase two-dimensional LC-MS was used for the shotgun proteomics of bronchoalveolar lavage (BAL) fluids from both mice and humans with Aspergillus fumigatus infection. Several proteins from Aspergillus fumigatus were detected, including thioredoxin reductase GliT. The protein has low sequence homology to mouse and human proteins and was found in both mice and human BAL samples, showing its great potential as a biomarker.
... An antibody fragment (MS112-IIB1) directed against the Chitin ring formation 2 (Crf2) protein of A. fumigatus was initially developed for diagnosis purposes (Schütte et al., 2009). Encoded by CRF1 gene, the Crf protein family contains three protein variants: Crf2 protein, amino acids (333 aa) long, Asp f9 protein (292 aa), first identified as an allergen using serum from Aspergillus-allergic patients (Crameri and Blaser, 1996), and Crf1 (395 aa, also named Crh5). Crf1 protein is the only member of the Crf family with a predicted glycosylphosphatidylinositol (GPI) anchor (Arroyo et al., 2007), and has been shown to induce a cross-protection between A. fumigatus and C. albicans infection (Stuehler et al., 2011). ...
Aspergillus fumigatus is an airborne opportunistic fungal pathogen responsible for severe infections. Among them, invasive pulmonary aspergillosis has become a major concern as mortality rates exceed 50% in immunocompromised hosts. In parallel, allergic bronchopulmonary aspergillosis frequently encountered in cystic fibrosis patients, is also a comorbidity factor. Current treatments suffer from high toxicity which prevents their use in weakened subjects, resulting in impaired prognostic. Because of their low toxicity and high specificity, anti-infectious therapeutic antibodies could be a new alternative to conventional therapeutics. In this study, we investigated the potential of Chitin Ring Formation cell wall transglycosylases of A. fumigatus to be therapeutic targets for therapeutic antibodies. We demonstrated that the Crf target was highly conserved, regardless of the pathophysiological context; whereas the CRF1 gene was found to be 100% conserved in 92% of the isolates studied, Crf proteins were expressed in 98% of the strains. In addition, we highlighted the role of Crf proteins in fungal growth, using a deletion mutant for CRF1 gene, for which a growth decrease of 23.6% was observed after 48 h. It was demonstrated that anti-Crf antibodies neutralized the enzymatic activity of recombinant Crf protein, and delayed fungal growth by 12.3% in vitro when added to spores. In a neutropenic rat model of invasive pulmonary aspergillosis, anti-Crf antibodies elicited a significant recruitment of neutrophils, macrophages and T CD4 lymphocytes but it was not correlated with a decrease of fungal burden in lungs and improvement in survival. Overall, our study highlighted the potential relevance of targeting Crf cell wall protein (CWP) with therapeutic antibodies.
... For the above reasons, the aim of the present study was to identify novel melanocyte autoantigens in vitiligo using the technique of phage-display based on the pJuFo cloning system (18,19). This technology has been used to identify allergens that bind to human IgE Ab's using yeast and fungal cDNA phagedisplay libraries (20)(21)(22). The strategy permits both the expression of cDNA libraries and the covalent attachment of the expressed products as Fos-fusion proteins on the surface of filamentous phage particles, thus allowing the selective enrichment of phage-displaying IgG peptides in rounds of biopanning. ...
... Most described libraries are in the range of 10 6 -10 9 variants, but larger libraries have also been described reaching over 10 10 members (Vaughan et al., 1998). Protein classes for which libraries have been constructed and functionally been displayed on phage include for example cytokines, receptors, enzymes, protease inhibitors, antibody fragments, DNA binding proteins, cDNA encoded proteins (Crameri and Blaser, 1996) and several different peptides (Clackson and Wells, 1994;Benhar, 2001). ...
... Here, the cDNAs are cloned as C-terminal fusions to the Fos leucine zipper domain and then tethered to the phage via a disulfide-stabilized interaction with the phage-displayed Jun-glIIp fusion (pJuFo system). Crameri et al. used pJuFo to display a cDNA library from Aspergillus fumigatus and cloned fungal allergens by binding to IgE from allergic individuals [25,26]. Screening of a pJuFo cDNA library from human lymphocytes against HIV-1 reverse transcriptase identified cellular 13-actin as a virus-host interaction [27]. ...
Knowing the sequence of a gene does not mean knowing its function. Although, information stored at the DNA level can be used to predict biological processes, proteins are the final executors of the various response programs of a cell. Transient information, like posttranslational modifications or interactions among proteins, cannot be deduced from DNA sequences. The rapid accumulation of large amounts of DNA sequence data in genomics projects has led to an increasing demand for powerful tools to analyze proteins and their behaviour at a large scale. This review aims to compare different technologies used for identification of interacting proteins and discusses recent developments in the field of high-throughput protein-protein interaction mapping.
... This system has been successfully used to identify allergen proteins: phage display cDNA libraries of biological materials that cause the allergy were screened, to identify the protein(s) that induce the allergic reaction. The baits in these screens were IgE antibodies of the patients (Crameri and Blaser, 1996). In another approach, a phage display phagemid vector was designed that contains a removable marker cassette (lox sites-flanked β lactamase gene) between the cloning site and gIII. ...
Filamentous bacteriophages contain a circular single‐stranded deoxyribonucleic acid (ssDNA) genome packaged into long filaments. These phages do not reproduce by lysing bacteria; instead, they are secreted into the environment without killing the host. Well‐studied Escherichia coli K12‐infecting Ff phages (f1, fd or M13) always replicate episomally; however a growing number of ‘lysogenic’ or chromosomally integrated filamentous phages of Gram‐negative bacteria are being discovered. The ‘lysogens’ can be induced; however phage reproduction does not require genome excision from bacterial chromosome and does not lyse the host cells. Some filamentous phages enhance the virulence of their host organisms, the most striking example being the CTXφ of Vibrio cholerae , which encodes cholera toxin. E. coli Ff phages are the workhorse of phage display technology, whose most notable ‘products’ are therapeutic recombinant antibodies. Ff are also being used in nanotechnology as templates for assembly of nanostructures, which has already led to their incorporation into a working nanobattery.
Key Concepts
Filamentous bacteriophages are long filaments (6–7 nm×>500 nm) that contain a single‐stranded circular DNA genome.
Filamentous bacteriophages replicate via a rolling circle mechanism, one strand at a time.
Filamentous bacteriophages do not lyse the cells; they are released by secretion, using a dedicated filamentous phage assembly secretion system.
Filamentous phage secretion‐assembly requires the proton‐motive force and ATP.
Some filamentous phages replicate exclusively as episomes, while others also integrate their genomes into the host chromosome, forming a lysogen. Induction of the lysogen does not result in cell lysis.
Ff filamentous phages of E. coli (f1, M13 and fd) have been used interchangeably as vectors and helper phages in DNA sequencing, as a protein display platform in phage display technology and as a template for assembly of nanostructures in nanotechnology.
... The allergenic significance of various airborne Aspergillus species like A fumigatus, A flavus, A niger, A nidulans, A versicolor and A oryzae has been extensively studied (Aggarwal et al. 2000;Kurup and Banerjee 2000;Simon-Nobbe et al. 2008;Shen et al. 1999). A number of A fumigatus allergens have been cloned from cDNA/phage display libraries, characterized and purified as recombinant proteins (Crameri and Blaser 1996;Crameri et al. 2006;Vailes et al. 2001). Alkaline-and vacuolar-serine proteases have been identified as major allergens of A flavus (Shen et al. 1999(Shen et al. , 2007Kurup and Banerjee 2000). ...
Aspergillus-derived inhalant allergens play an important role in the etiology of allergic respiratory diseases. In the present study, we investigated the allergenic potential of Aspergillus tamarii, quantified its airborne content, identified its major/minor allergens, evaluated heterogeneity of patients' IgE response to its allergens and cross-reactivity of its allergens with other Aspergillus allergens. Skin prick tests with A tamarii extract were performed on 300 patients of bronchial asthma/allergic rhinitis and 20 healthy volunteers. Sixty-six patients (22%) elicited positive cutaneous reactions to A tamarii extract. Only one of the 20 non-allergic healthy volunteer showed a mild positive cutaneous reaction. Allergen-specific IgE levels increased with increase in patients' cutaneous response (0% in negative to 100% in 3+/4+). The skin positivity and allergen-specific IgE levels were significantly higher in patients compared to healthy volunteers (P>0.05). However, no differences were found for these two parameters among patients of bronchial asthma, allergic rhinitis and bronchial asthma with allergic rhinitis. The airborne A tamarii allergen content was highest in February and October. A tamarii extract revealed at least 22 proteins (13.3-120 kDa). Seventeen of these proteins bound patients' IgE with six being major allergens (13.3, 23, 25, 34, 39.5, 43 kDa). Three major allergens (13.3, 34, 43 kDa) were found to cross-react with A flavus and one (34 kDa) with A niger. Our results revealed that A tamarii allergen(s) are present in the air, which might serve as important inhalant allergens in IgE-mediated allergic respiratory diseases.
... These results prove the integrity of the bispecific phagemid vector pHEN3+9 ( indicates that all other classes of proteins, as described by Smith and Petrenko (1997), could also be functionally displayed using the bispecific phagemid vector pHEN3+9. In conclusion, the immobilization of all kind of protein libraries, like genomic libraries (Jacobsson et al. 1995), peptide libraries (Cwirla et al. 1990), different kinds of antibody libraries (Hoogenboom et al. 1991; Griffiths 1993; Winter et al. 1994), cDNA libraries (Crameri et al.1994; Crameri and Blaser 1996) and even full-length cDNA libraries using the Fos/Jun-system (Crameri and Suter, 1993; Palzkill et al. 1998) became possible and might be adapted to automated screening systems as e.g. the envisaged Protein Nanochip (Fig.I-9). ...
Aachen, Techn. Hochsch., Diss., 2005 (Nicht für den Austausch).
... This allows selective enrichment of phage displaying proteins able to interact with a ligand (IgE) immobilized on a solid support whereas noninteracting phage remain in the liquid phase. Several allergens from A. fumigatus have been successfully cloned using the pJuFo system (Crameri and Blaser, 1996). Here we report the application of the pJuFo cloning system to enrich allergen encoding cDNA from a M. furfur phage surface display library. ...
The yeast Malassezia furfur, also known as Pityrosporum orbiculare (ovale), is part of the normal microflora of the human skin but has also been associated with different skin diseases including atopic dermatitis. More than 50% of atopic dermatitis patients have positive skin test and specific IgE to M. furfur extracts; however, the pathophysiologic role of these IgE-mediated reactions in the development of the disease remains unknown. The yeast is able to produce a wide panel of IgE-binding proteins, variably recognized by sera of individual patients. In order to assess the contribution of individual components to the disease, highly pure allergen preparations are required. We have cloned M. furfur allergens from a cDNA library displayed on the phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. Phage displaying IgE-binding proteins were selectively enriched from the library using IgE from a M. furfur-sensitized atopic dermatitis patient as a ligand. We were able to identify five different inserts coding for IgE-binding polypeptides. Three of the sequenced cDNA encode incomplete gene products with molecular masses of 21.3 kDa (MF 7), 14.4 kDa (MF 8), and 9.7 kDa (MF 9), respectively, having no sequence similarity to known proteins. The other two cDNA encode allergens of 18.2 kDa (Mal f 5) and 17.2 kDa (Mal f 6). Mal f 5 shows significant homology to M. furfur allergens Mal f 2, Mal f 3 and an Aspergillus fumigatus allergen Asp f 3. Mal f 6 has significant homology with cyclophilin. All of the recombinant polypeptides were capable of binding serum IgE from atopic dermatitis patients in immunoblotting experiments. The availability of pure recombinant M. furfur allergens will allow the careful investigation of the role of IgE-binding proteins in atopic dermatitis.
Food allergy is a serious food safety problem worldwide, and the investigation of food allergens is the foundation of preventing and treating them, but relevant knowledge is far from sufficient. With the advent of the "big data era", it has been possible to investigate food allergens by high-throughput methods, proposing the concept of allergomics. Allergomics is the discipline studying the repertoire of allergens, which has relatively higher throughput and is faster and more sensitive than conventional methods. This review introduces the basis of allergomics and summarizes its major strategies and applications. Particularly, strategies based on immunoblotting, phage display, allergen microarray, and bioinformatics are reviewed in detail, and the advantages and limitations of each strategy are discussed. Finally, further development of allergomics is predicted. This provides basic theories and recent advances in food allergomics research, which could be insightful for both food allergy research and practical applications.
Fungi form a large kingdom with more than 1.5 million species. Fungal spores are universal atmospheric components and are generally recognized as important causes of allergic disorders, including allergic rhinitis, allergic rhinosinusitis, asthma, and allergic bronchopulmonary aspergillosis. The 4 genera which have the closest connection with allergic disorder are Cladosporium, Alternaria, Aspergillus and Penicillium. The cDNA sequences of many fungi allergens and the amino acids involved in their immunoglobulin E binding and T-cell activation have already been elucidated. Until now, 111 allergens from 29 fungal genera have been approved by the International Allergen Nomenclature Sub-committee. This review mainly focuses on the biochemical characteristics and allergenic activity of important allergens from common environmental fungi.
Es wird immer noch angenommen, dass Kinder mit cystischer Fibrose (CF) mit histoanatomisch normalen Lungen zur Welt kommen [30]. Zweifelsohne beginnt sich aber der pulmonale Befall bei den meisten Patienten immunologisch und funktionell bereits in den ersten Lebensmonaten zu manifestieren. Im Vordergrund steht bereits in diesem Alter einerseits die Imbalance zwischen Proteasen (Leukozytenelastase) und Antiproteasen (α1-Antitrypsin etc.) im Schleimhautfilm des Bronchialtraktes [4]. Andererseits ist lungenphysiologisch bei vielen Säuglingen eine pulmonale Überblähung festzustellen, ein Befund, welcher klinisch über Monate verborgen bleiben kann, und vom Genotyp abhängig zu sein scheint [13]
Incidence of fungal infections has increased alarmingly in past few decades. Of the fungal pathogens, the Aspergillus fumigatus has been a major cause of allergic bronchopulmonary aspergillosis (ABPA) which has five main stages - the acute, remission, exacerbation, glucocorticoid dependent and fibrotic stage. The diagnosis of ABPA remains difficult due to its overlapping clinical and radiological features with tuberculosis and cystic fibrosis. From past few decades, the crude fractions of A. fumigatus have been used for immunodiagnosis of ABPA. Most of the detection kits based on crude fractions of A. fumigatus are quite sensitive but have low specificity. Till date 21 known and 25 predicted allergens of A. fumigatus have been identified. Of these allergens, only five recombinants (rAsp f1-f4 and f6) are commercially used for diagnosis of allergic aspergillosis. Remaining allergens of A. fumigatus have been restricted for use in specific diagnosis of ABPA, due to sharing of common antigenic epitopes with other allergens. Complete sequencing of A. fumigatus genome identified 9926 genes and the reports on the proteome of A. fumigatus have shown the presence of large number of their corresponding proteins in the pathogen. The analysis of immunoproteomes developed from crude fractions of A. fumigatus by IgG/IgE reactivity with ABPA patients and animal sera have identified the panel of new antigens. A brief description on the current status of A. fumigatus antigens is provided in this review. The implementation of advance recombinant expression and peptidomic approaches on the A. fumigatus antigens may help in the selection of appropriate molecules for the development of tools for more specific early diagnosis of ABPA, and desensitization therapies for patients of allergic disorders.
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Zusammenfassung. Die Klonierung, Sequenzierung und Produktion von hochreinen Allergenen bietet die Möglichkeit, perfekt standardisierte Allergenpräparate herzustellen. Die Entwicklung eines neuen Klonierungssystems, das auf filamentösen Phagen basiert, führte zu einer schnellen Isolierung und Charakterisierung von Aspergillus fumigatus-Allergenen. Die auf diesem Weg rekombinant hergestellten Proteine wurden serologisch und klinisch geprüft und ihr routinemä-ßiger Einsatz im ImmunoCAP-System evaluiert. Es gelang eine quantitative Übereinstimmung zwischen Hauttestergebnissen und Serologie nachzuweisen, welche das Potential rekombinanter Allergene in der Diagnostik allergischer Krankheiten aufzeigt. Darüber hinaus trägt die Charakterisierung der Pilzallergene wesentlich zum Verständnis der moiekularen Natur der allergieauslösenden Komponenten bei Zum jetzigen Zeitpunkt können, abgesehen von Proteinen mit unbekannten biologischen Funktionen, die Pilzallergene in zwei Klassen eingeteilt werden: 1. Spezies-spezifische sezcrnierte Allergene und 2. cytoplasmatische, hoch konservierte Proteine. Diese letztgenannten Pilzallergene zeigen auch zu Proteinen aus phylogenetisch weit entfernten Organismen weitreichende Sequenzhomologien. Neben der daraus zu erwartenden IgE-Kreuzreaktivität findet man in einigen Fällen auch eine Kreuzreaktivität mit den homologen humanen Proteinen, was auf Autoimmunreaktionen, bei Pilzalleigien hindeutet.
Aspergillus fumigatus ribotoxin Asp f 1 is a major allergen with IgE binding activity to serum of a majority of patients with allergic bronchopulmonary aspergillosis (ABPA). The IgE binding epitopes or the T-cell stimulatory peptides of this molecule have not been studied. In the present investigation, we have synthesized linear decapeptides spanning the whole molecule of Asp f 1 and analyzed their IgE binding properties. We have also synthesized peptides based on their possible T-cell stimulatory properties and studied the stimulation of peripheral blood mononuclear cells from ABPA patients and normal controls. Several peptides demonstrated distinct IgE antibody binding response against sera from ABPA patients and proliferative response against peripheral blood mononuclear cells from the patients. From the results, it can be concluded that the carboxy-terminal region of Asp f 1 representing amino acid residues 115–149 involved in both humoral and cell mediated immunoresponses in ABPA.
Phage display involves the production and screening of large numbers of random peptide sequences of a specific length expressed on the surface of phage particles. This approach provides a powerful tool to probe the molecular basis of many biological processes, including host–parasite interactions. Phage display libraries have been used to study the binding specificity of numerous peptides and protein domains. Practical applications include the identification of peptide sequences that bind with high affinity to antibodies, enzymes or receptors, and that may serve as diagnostics and vaccine or drug candidates. Here, David Jefferies outlines the concept of phage display and summarizes recent developments in the field, with emphasis on those that may be of interest to parasitologists.
A fragment of antigen binding (Fab) surface display system was developed using a glycoengineered Pichia pastoris host strain genetically modified to secrete glycoproteins with mammalian mannose-type Man(5)GlcNAc(2) N-linked glycans. The surface display method described here takes advantage of a pair of coiled-coil peptides as the linker while using the Saccharomyces cerevisiae Sed1p GPI-anchored cell surface protein as an anchoring domain. Several Fabs were successfully displayed on the cell surface using this system and the expression level of the displayed Fabs was correlated to that of secreted Fabs from the same glycoengineered host in the absence of the cell wall anchor. Strains displaying different model Fabs were mixed and, through cell sorting, the strain displaying more expressed Fab molecule or the strain displaying the Fab with higher affinity for an antigen was effectively enriched by FACS. This novel yeast surface display system provides a general platform for the display of Fab libraries for affinity and/or expression maturation using glycoengineered Pichia.
In filamentous fungi, secondary metabolism is often linked with developmental processes such as conidiation. In this study
we analyzed the link between secondary metabolism and conidiation in the main industrial producer of the β-lactam antibiotic
penicillin, the ascomycete Penicillium chrysogenum. Therefore, we generated mutants defective in two central regulators of conidiation, the transcription factors BrlA and StuA.
Inactivation of either brlA or stuA blocked conidiation and altered hyphal morphology during growth on solid media, as shown by light and scanning electron microscopy,
but did not affect biomass production during liquid-submerged growth. Genome-wide transcriptional profiling identified a complex
StuA- and BrlA-dependent regulatory network, including genes previously shown to be involved in development and secondary
metabolism. Remarkably, inactivation of stuA, but not brlA, drastically downregulated expression of the penicillin biosynthetic gene cluster during solid and liquid-submerged growth.
In agreement, penicillin V production was wild-type-like in brlA-deficient strains but 99% decreased in stuA-deficient strains during liquid-submerged growth, as shown by high-performance liquid chromatography (HPLC) analysis. Thus,
among identified regulators of penicillin V production StuA has the most severe influence. Overexpression of stuA increased the transcript levels of brlA and abaA (another developmental regulator) and derepressed conidiation during liquid-submerged growth but did not affect penicillin
V productivity. Taken together, these data demonstrate an intimate but not exclusive link between regulation of development
and secondary metabolism in P. chrysogenum.
Phage display has been extensively used to study protein-protein interactions, receptor- and antibody-binding sites, and immune responses, to modify protein properties, and to select antibodies against a wide range of different antigens. In the format most often used, a polypeptide is displayed on the surface of a filamentous phage by genetic fusion to one of the coat proteins, creating a chimeric coat protein, and coupling phenotype (the protein) to genotype (the gene within). As the gene encoding the chimeric coat protein is packaged within the phage, selection of the phage on the basis of the binding properties of the polypeptide displayed on the surface simultaneously results in the isolation of the gene encoding the polypeptide. This unit describes the background to the technique, and illustrates how it has been applied to a number of different problems, each of which has its neurobiological counterparts. Although this overview concentrates on the use of filamentous phage, which is the most popular platform, other systems are also described.
Persistierende Infektionen mit Borrelia burgdorferi sensu lato, dem Erreger der Lyme Borreliose (LB), als auch mit Chlamydia pneumoniae, einem häufigen Atemwegspathogen, stellen ein erhebliches Gesundheitsrisiko dar. Für beide Pathogene ist bisher keine hinreichende Serologie verfügbar, was u.a. darauf zurückzuführen ist, dass beide Bakterien über spezielle Strategien verfügen, mit deren Hilfe sie die Immunantwort des Wirtes umgehen können. Die Diagnose einer Borrelieninfektion wird neben den klinischen Befunden hauptsächlich durch den Nachweis gebildeter Antikörper geführt. Die Zuverlässigkeit dieses Verfahrens ist durch die unzureichende Sensitivität und Spezifität der gegenwärtig erhältlichen Tests eingeschränkt. Im ersten Teil dieser Arbeit wurden deshalb mittels "Phage surface display" neue Borrelienantigene gesucht und deren immunogene Eigenschaften charakterisiert. - Genomische Phagenbibliotheken von B. afzelii, B. burgdorferi und B. garinii wurden erstellt. Über Affinitätsanreicherung mit IgG-Antikörpern von LB Patienten wurden neben dem bereits beschriebenen Antigen BBK32 acht weitere, neue Proteine selektiert. Davon zeigte das ribosomale Protein L25 ebenfalls antigene Eigenschaften. In einem mit 80 Seren von LB Patienten und 75 Seren von Kontrollpersonen etablierten ELISA, erreichte L25 eine Sensitivität von 23% und eine Spezifität von 99%, was in Kombination mit anderen Antigenen, für dessen Eignung zur Serodiagnose spricht. Die bisherigen Kenntnisse über Chlamydienantigene sind limitiert, was die Entwicklung von sensitiven und spezifischen serologischen Tests erschwert. Ein großer Nachteil der heutigen C. pneumoniae Serodiagnose besteht darin, dass persistierende Infektionen nicht von einer Seronarbe unterschieden werden können. Im zweiten Teil dieser Arbeit wurden daher mittels "Immunoproteomics" neue Chlamydienantigene identifiziert, die einerseits eine sensitive und spezifische Serologie zulassen, und andererseits die Differenzierung zwischen vergangenen und persistenten Infektion ermöglichen könnten. - Über Immunoblots nach 2D-SDS-PAGE und massenspektrometrischer Analyse IgG reaktiver Antigenspots wurden 31 Hauptantigene von C. pneumoniae identifiziert, von denen 15 bisher unbekannte Antigene darstellen. Eine differentielle Analyse der Spotintensität ergab, dass 8 Antigene eine erhöhte Reaktivität mit Seren von mutmaßlich persistent infizierten Spendern (n=19) aufwiesen und 4 Antigene präferentiell mit Seren nicht-persistent infizierter Spender (n=15) reagierten. Mit dieser Analyse wurde eine erste Grundlage für die Diagnose persistenter Infektionen auf serologischer Basis geschaffen. Im dritten Teil dieser Arbeit wurde untersucht, ob die durch C. pneumoniae induzierte Aktivierung von T Gedächtniszellen einen geeigneten Parameter zur Identifizierung persistenter Infektionen mit C. pneumoniae darstellt. - Mittels Multiparameter-Durchflusszytometrie wurde die Chlamydien induzierte Aktivierung von T Zellen in verschiedenen Spendern analysiert. Nach Inkubation von isolierten PBMC mit C. pneumoniae oder deren Zytosol konnten bis zu 0,12% aktivierte CD4+ T-Zellen detektiert werden, während keine Aktivierung von CD8+ T-Zellen nachweisbar war. Die Charakterisierung der CD4+ T Zellen hinsichtlich ihrer Stabilität und Zytokinproduktion erlaubte deren Klassifizierung als T Gedächtniszellen. Da bei 9 von 17 Spendern mit einer mutmaßlich persistenten Infektion CD4+ T Gedächtniszellen nachgewiesen wurden, während dies nur bei 1 von 10 nicht-persistent infizierten Spendern der Fall war, könnte der Nachweis von T Gedächtniszellen als Parameter für die Diagnose persistenter Infektion mit C. pneumoniae herangezogen werden. Bisher ist es nicht geklärt, ob C. pneumoniae Peptidoglykan (PGN) synthetisieren. Die Erkennung von PGN durch das angeborene Immunsystem spielt sowohl bei der Aktivierung der T Zell vermittelten Immunantwort, als auch bei der Produktion von Antikörpern eine entscheidende Rolle. Im Falle der Abwesenheit von PGN in Chlamydien könnte man vermuten, dass während einer Chlamydieninfektion lediglich suboptimale Immunantworten induziert werden, die eine Persistenz der Bakterien begünstigen könnten. Im vierten Teil dieser Arbeit wurde die Transkription chlamydialer Gene untersucht, die mit der Synthese von PGN assoziiert werden. - Mittels einer neu etablierten hochsensitiven real-time PCR Methode konnte gezeigt werden, dass alle der 9 untersuchten PGN-assoziierten Gene während des Infektionszyklus von C. pneumoniae transkribiert werden, was auf eine aktive PGN-Biosynthese hinweist. Die enge zeitliche Überlappung der Transkription mit dem frühen Vermehrungsprozess von C. pneumoniae und deren anschließende rasche Abnahme spricht für die Präsenz von PGN, sowie für dessen Rolle in chlamydialen Retikularkörperchen, nicht aber in Elementarkörperchen. Zusammenfassend lässt sich sagen, dass diese Arbeit zu einem besseren Verständnis der adaptiven Immunantwort gegen B. burgdorferi sensu lato und C. pneumoniae beiträgt. Die hier präsentierten Daten stellen nicht nur eine Basis für die Überwindung der gegenwärtigen Defizite diagnostischer Anwendungen dar, sondern besitzen auch potentielle Bedeutung für Impf- und Behandlungsstrategien. Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis (LB), and Chlamydia pneumoniae, a common cause of respiratory tract infections, are able to establish persistent infections that can be harmful to the host. Both pathogens lack an adequate serology, which appears to be linked to unique host immune responses associated with different immune evasion strategies of these bacteria. Beside the clinical manifestations, the diagnosis of Borrelia infections is commonly based on serological testing, which has major shortcomings concerning sensitivity and specificity. The performance of currently available serological tests might be improved by using more sensitive and more specific Borrelia antigens. In the first part of this thesis, novel Borrelia antigens were indentified by phage display technology and characterized with regard to their diagnostic value. - Genomic phage surface display libraries were generated from B. afzelii, B. burgdorferi and B. garinii. Affinity selection against IgG from LB patients revealed nine different Borrelia proteins, including the well established antigen BBK32. Another identified protein, the ribosomal protein L25, was demonstrated to be antigenic by enzyme linked immunosorbent assay using sera from 80 LB patients and 75 controls. The specificity and sensitivity of the antigen was 99% and 23%, respectively, qualifying L25 as a useful antigen when combined with others. The development of a sensitive and specific C. pneumoniae serodiagnosis remains difficult because of the limited knowledge about appropriate antigens. To date, C. pneumoniae serology cannot distinguish between past and persistent infection, which represents a major shortcoming. In the second part of this thesis, novel C. pneumoniae antigens were identified and characterized using immunoproteomics. Further, it was evaluated whether the identified antigens can be used to discriminate past from persistent infections. - Using a proteomic approach combined with immunoblotting, we identified 31 major C. pneumoniae antigens, including 15 novel Chlamydia antigens that showed reactivity towards IgG antibodies. When analyzing the intensity of immunoreactive spots among 19 donors with and 15 donors without evidence for persistent infection, we identified 8 persistence associated antigens and 4 antigens that were associated with non persistent infections. These data represent a basis for a serodiagnosis allowing the identification of persistently infected individuals. In the third part of this thesis, the value of C. pneumoniae induced T cell responses for discriminating persistent from non-persistent infections was investigated. - C. pneumoniae induced T cell responses in different donors were analyzed by flow cytometric multiparameter analysis. After stimulation of PBMC with whole bacteria or cytosol, up to 0.12% activated CD4+ T cells were detected, while no CD8+ T cell activation was found. The characterization of the activated CD4+ T cells with regard to their cytokine production and their stability, argued for memory CD4+ T cells primed during persistent infections. Indeed, C. pneumoniae induced memory CD4+ T cell responses were detected in 9 out of 17 donors with evidence, but only in 1 out of 10 donors without evidence for persistent infections, which is of potential interest for diagnostic applications. Innate immune recognition of peptidoglycan (PGN) has a key role in the onset of antigen specific T cell responses and subsequent antibody production. Thus, infections with Chlamydia, a pathogen that lack detectable amounts of PGN, might provoke suboptimal adaptive immune responses possibly contributing to chlamydial persistence. Since the synthesis of chlamydial PGN is being debated, in the fourth part of the thesis, the transcription of chlamydial genes associated with PGN synthesis was analyzed. Highly sensitive and specific real time PCRs were developed to analyze the transcript levels of nine chlamydial genes associated with PGN synthesis. All genes were strongly upregulated during the C. pneumoniae infection cycle in HEp 2 cells arguing for an active PGN synthesis pathway. The transcription kinetics of these genes closely coincided with the early replication phase of C. pneumoniae indicating a role for the presence of PGN in dividing chlamydial reticulate bodies (RB). Since the transcription was markedly decreased at a time when the RB differentiated back into elementary bodies (EB), PGN is likely not, or only in low amounts, present in Chlamydia EB. In summary, this thesis contributes to the understanding of acquired immune responses against B. burgdorferi sensu lato and C. pneumoniae with regard to diagnostic applications. The presented data provide not only a platform for the development of new diagnostic tools that possibly overcome the current limitations but also might be of interest for vaccination and treatment strategies.
The advent of the serological identification of antigens by procedures such as cDNA cloning and recombinant protein expression has allowed the direct molecular definition of immunogenic proteins. The phage-display technology provides several advantages over conventional immunoscreening procedures based on plasmid or lambda-phage cDNA libraries. So far, attempts to display open reading frames, such as those encoded by cDNA fragments, on filamentous phages have not been very successful. We managed to develop a strategy based on “folding reporters” which allows filtering out open reading frames from DNA and displaying them on filamentous phages in such a way that they are amenable to subsequent selection or screening.
Once the cDNA library of interest is created, phage-display technology is used for selection of novel putative antigens; these are then validated by printing isolated protein on microarray and screening with patients’ sera.
We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.
From an Aspergillus fumigatus complementary deoxyribonucleic acid (cDNA) library displayed on phage surface, an allergen formally termed rAsp f 3 was cloned. The open-reading frame of the cloned gene for the allergen encodes a protein of 168 amino acids with a predicted molecular mass of 18.5 kD, showing 36% identity and 58% similarity to two peroxisomal membrane proteins of Candida boidinii. Recombinant Asp f 3 was expressed as a [His]6-tagged fusion protein in Escherichia coil at yields of 30 mg/L, and was purified by Ni(2+)-chelate chromatography. In an enzyme-linked immunosorbent assay (ELISA), serum IgE antibody reactivity to rAsp f 3 could be detected in 72% of 89 individuals sensitized to A. fumigatus, demonstrating that the protein represents a major allergen of the mold. IgE specific to rAsp f 3 and the two recombinant Candida proteins was further demonstrated by IgE-immunoblot analysis. IgE binding to rAsp f 3 could be inhibited in the ELISA by adding either of the recombinant Candida peroxisomal proteins to sera containing IgE directed against Asp f 3. Taken together, these observations prove that the Asperigillus allergen and the two Candida proteins share IgE-binding epitopes.
Members of the genus Aspergillus are opportunistic pathogens for cold- and warm-blooded animals. They are associated with an impressive spectrum of diseases in humans, ranging from benign colonization of the lung to life-threatening diseases such as invasive aspergillosis or allergic bronchopulmonary aspergillosis (ABPA). Aspergillus fumigatus is the etiological agent identified in most of the Aspergillus-related human diseases and is therefore of particular clinical relevance. A major problem in the diagnosis of A. fumigatus-related complications arises from the lack of standardized fungal extracts. The allergenicity of commercial A. fumigatus extracts depends on many factors including the strain used, growth conditions, harvesting and extraction procedures hampering the development of diagnostic reagents suitable for the discrimination between A. fumigatus-related diseases. Molecular DNA/RNA technologies and biotechnological procedures allow cloning, characterization and production of large amounts of single, highly pure allergens. The development of phage surface display technologies for cloning cDNAs has speeded up the isolation of candidate cDNAs encoding allergens. Screening of an A. fumigatus cDNA library displayed on the surface of phage M13 allowed characterization and production of a panel of allergens identified as proteins with known biological function or as allergens without sequence homology to known proteins. They belong to two functional categories: secreted and cytoplasmic proteins. Secreted allergens are recognized by serum IgE of A. fumigatus-sensitized individuals with or without ABPA, whereas nonsecreted allergens are exclusively recognized by serum IgE of ABPA patients. The dissection of IgE-mediated immune responses to single A. fumigatus allergens allows a discrimination between ABPA and A. fumigatus sensitization with high specificity and sensitivity demonstrating the power of recombinant allergens for the differential diagnosis of A. fumigatus-related pulmonary complications.
Cloning, sequencing and production of highly pure recombinant allergens allows to produce perfectly standardised allergen preparations. The development of a new cloning system based on filamentous phage allowed the fast isolation and characterisation of allergens from the fungus Aspergillus fumigatus. The produced recombinant allergens were tested in serological and clinical studies as well as for their performance for routine assessments in the ImmunoCAP-system. Thereby, a perfect correlation between skin test results and serology was found showing the potential of recombinant allergens for the diagnosis of allergic diseases. Moreover, the characterisation of fungal allergens substantially contributes to our understanding of the molecular nature of proteins involved in the elicitation of allergic reactions. Apart from allergenic proteins with unknown biological function, fungal allergens can be subdivided into two classes: 1. Species-specific, secreted proteins and 2. cytoplasmic, even in phylogenetically distant organisms, well conserved proteins. These fungal allergens show extended sequence similarity, a high level of IgE cross-reactivity and in some cases also cross-reactivity with homologous human proteins indicating autoimmune reactions involved in fungal allergy.
The in vitro diagnostic value of recombinant allergens depends on the correlation between allergen specific IgE in serum and ability of the protein to elicit immediate type allergic reactions in sensitized individuals. Objective The study was carried out to evaluate the reliability of recombinant Asp f 3 (rAsp f 3)-based serological determinations in ELISA and ImmunoCAP compared to skin-test responses in A. fumigatus sensitized subjects.
Patients suffering from allergic bronchopulmonary aspergillosis (ABPA, n=11) or allergic asthma with A. fumigatus sensitization (n =8) and healthy control subjects (n=4) were investigated. Intradermal skin tests (IDT) and allergen-specific serology were performed using rAsp f 3 protein.
All 11 patients with ABPA and five out of eight A. fimigatus-sensitized asthmatics without ABPA exhibited a type I skin reaction. All rAsp f 3 skin test positive subjects showed relevant rAsp f 3-specific serum IgE levels compared with IDT-negative individuals who scored below the rAsp f 3-ImmunoCAP cut-off values of < 0.35 kU(A)/L. Analyses of rAsp f 3-specific immunoglobulins revealed significantly higher serum levels of IgG, IgG1, IgG4 and IgE antibodies in patients with ABPA compared with A. fumigatus-sensitized asthmatics and healthy controls.
rAsp f 3 represents a major allergen of A. fumigatus. It is recognized by 84% of asthmatic individuals sensitized to the fungus and elicits specific type I skin reactions. The skin test outcome strictly depends on the presence of rAsp f 3-specific IgE and the lack of false positive or false negative results comparing skin test outcome with specific serum IgE levels determined by ELISA or rAsp f 3-ImmunoCAP suggests the possibility to rely on serological data obtained with recombinant allergens to diagnose sensitization to A. fumigatus.
Allergic bronchopulmonary aspergillosis (ABPA), a severe pulmonary complication caused by Aspergillus fumigatus, is considered a complex clinical syndrome with defined serological, pathological radiological and clinical features. The diagnosis of ABPA is often difficult because of several overlapping clinical and laboratory findings shared between asthma with sensitization to A. fumigatus and ABPA, but essential for treatment to prevent severe deterioration of pulmonary function. We have cloned A. fumigatus allergens from a cDNA library displayed on phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. The single recombinant allergens were used to assess the immunological response in representative groups of A. fumigatus-sensitized asthmatics with or without ABPA and healthy controls. The allergens rAsp f 1, a 16.9 kDa ribotoxin, rAsp f 3, a 18.5 kDa peroxisomal protein, and rAsp f 6, a 23 kDa manganese superoxide dismutase, were identified as proteins with known biological function and rAsp f 4, a 30 kDa allergen, lacks sequence homology to known proteins. The secreted ribotoxin rAsp f 1 and rAsp f 3 are recognized by serum IgE of A. fumigatus-sensitized asthmatics with or without ABPA, whereas the non-secreted manganese superoxide dismutase rAsp f 6 and the rAsp f 4 allergen are exclusively recognized by serum IgE of ABPA patients. The dissection of IgE-mediated immune responses to single recombinant A. fumigatus allergens in asthmatic patients allow a discrimination between ABPA and A. fumigatus sensitization with high specificity (100%) and sensitivity (90%).
Aspergillus-derived enzymes are used in dough improvers in bakeries. Some of these enzymes are identified as causing IgE-mediated sensitization in up to 25% of bakers with workplace-related symptoms.
The aim of this study was to compare the frequency of sensitization to Aspergillus xylanase, cellulase, and glucoamylase with the sensitization to alpha-amylase (Asp o 2) and to identify IgE-reactive proteins in enzyme preparations.
Sensitization to Aspergillus-derived enzymes and cross-reactivity were retrospectively studied by enzyme allergosorbent test (EAST) and EAST-inhibition experiments. IgE-reactive proteins were detected by electrophoretic separation and immunoblotting. Liquid chromatography with electrospray ionization mass spectrometry and Edman degradation of tryptic protein fragments were used for the biochemical identification of an unknown IgE-binding protein.
Twenty-three percent of 171 tested bakers had specific IgE to alpha-amylase, 8% reacted to glucoamylase, 13% reacted to cellulase, and 11% reacted to xylanase. Xylanase and cellulase preparations, each containing at least 6 different proteins, showed cross-reactivity in the range of 80%. The main IgE-binding protein in the xylanase preparation recognized in 7 of 8 xylanase-positive subjects was a protein of about 105 kd. This protein was identified as beta-xylosidase by peptide mass spectrometric fingerprinting. The identification was confirmed by matching 12 peptide sequences obtained by N-terminal and mass spectrometric sequencing to this protein.
Beta-Xylosidase from Aspergillus niger is an occupational allergen present in currently used baking additives, which causes sensitization in at least 4% of symptomatic bakers. According to the International Union of Immunological Societies nomenclature, we suggest the term Asp n 14 for this allergen.
The 37-kDa recombinant protein Asp f 2, encoding an allergen of Aspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 from Aspergillus nidulans and fibrinogen binding protein from Candida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereas A. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen of A. fumigatus exhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis of A. fumigatus-associated allergic diseases.
Functional genomics is facing the challenge of functionally identifying thousands of genes generated by the various genome projects during the past decade. Success will require novel high-capacity technologies that can cope with very large numbers of molecules. Here, Carl Borrebaeck reviews recent developments in molecular libraries and their role in rational gene identification.
Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy.
Peanut kernels contain many allergens able to elicit IgE-mediated type 1 allergic reactions in sensitized individuals. Sera from sensitized patients recognize variable patterns of IgE-binding proteins. The identification of the IgE-binding proteins of peanut extract would faciliate improvement of diagnostic and immunotherapeutic approaches as well as development of sensitive test systems for the detection of hidden peanut allergens present as additives in various industrial food products and the investigation of their stability during processing of food products.
We applied the pJuFo cloning system based on the phage surface display of functional cDNA expression products to clone cDNAs encoding peanut allergens. Sera (n = 40) of peanut-allergic individuals were selected according to case history, radioallergosorbent test and immunoblot analysis to demonstrate IgE binding towards the newly identified recombinant allergens.
In addition to the known allergens Ara h 1 and Ara h 2 we were able to identify four allergens with estimated molecular weights of 36, 16, 14.5 and 14 kDa. Three of them formally termed Ara h 4, Ara h6 and Ara h 7 show significant sequence similarities to the family of seed storage proteins and the fourth (Ara h 5) corresponds to the well-known plant allergen profilin. Immunoblotting of the six expressed recombinant allergens with 40 patients sera shows 14 individual recognition patterns and the following frequency of specific IgE binding: Ara h 1 was recognized by 65%, Ara h 2 by 85%, Ara h 4 by 53%, Ara h 5 by 13%, Ara h 6 by 38% and Ara h 7 by 43% of the selected sera.
All of the selected peanut-positive sera can detect at least one of the six identified recombinant allergens which can be used to establish individual patients' reactivity profiles. A comparison of these profiles with the clinical data will possibly allow a further insight into the relationship between clinical severity of the symptoms and specific IgE levels towards the six peanut allergens.
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.
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).
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.
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.
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.
Phage display is an advanced technology that can be used to characterize the interactions of antibody with antigen at the molecular level. It provides valuable data when applied to the investigation of IgE interaction with allergens. The aim of this rostrum article is to provide an explanation of the potential of phage display for increasing the understanding of allergen-IgE interaction, the discovery of diagnostic reagents, and the development of novel therapeutics for the treatment of allergic disease. The significance of initial studies that have applied phage display technology in allergy research will be highlighted. Phage display has been used to clone human IgE to timothy grass pollen allergen Phl p 5, to characterize the epitopes for murine and human antibodies to a birch pollen allergen Bet v 1, and to elucidate the epitopes of a murine mAb to the house dust mite allergen Der p 1. The technology has identified peptides that functionally mimic sites of human IgE constant domains and that were used to raise antiserum for blocking binding of IgE to the FcepsilonRI on basophils and subsequent release of histamine. Phage display has also been used to characterize novel peanut and fungal allergens. The method has been used to increase our understanding of the molecular basis of allergen-IgE interactions and to develop clinically relevant reagents with the pharmacologic potential to block the effector phase of allergic reactions. Many advances from these early studies are likely as phage display technology evolves and allergists gain expertise in its research applications.
Individual immunoprint patterns of sodium dodecylsulfate-polyacrylamide gel electrophoresis-separated Aspergillus fumigatus (Af) allergens/antigens were evaluated in 28 patients with allergic bronchopulmonary aspergillosis in stages II to V. It could be demonstrated that active disease (stage III) is characterized by a very strong IgE response against a variety of Af components, whereas the IgE antibody pattern in corticosteroid-treated patients who have demonstrated improvement is much weaker. In patients in remission (stage II), it is minimal. In contrast, many patients in stage V without corticosteroid treatment demonstrated a strong reactivity of IgE antibodies, indicating persisting active disease. The pattern of IgG antibodies with individual Af components resembles, in general, that of IgE antibodies; however, discrimination between different stages and between treated patients is much weaker. Our results indicate that a certain relationship between the different stages of allergic bronchopulmonary aspergillosis and Af immunoprint patterns exists.
Short ragweed allergenic extract has been studied by means of crossed radioimmunoelectrophoresis (CRIE) with the use of sera from 37 allergic patients and the relevant control sera. In this study 22 of 52 antigens, detectable in crossed immunoelectrophoresis (CIE) against polyspecific rabbit anti-ragweed IgG, were able to bind specific human IgE to their corresponding immunoprecipitates. This binding was semiquantified by comparison with the binding of a standard serum pool. Nine antigens were identified as important allergens, including the previously isolated components, AgE, AgK, and Ra6. Certain allergens (e.g., AgE, AgK, and Ag 31) bound IgE in almost all patients' sera, whereas others showed a bimodal distribution for sera of responder and nonresponder patients. The total CRIE score was found to correlate significantly both with ragweed-specific serum IgE antibody determined by RAST (rs = 0.88; p less than 0.001) and with total IgE level (rs = 0.55; p less than 0.01). Patient's CRIE scores to AgE also correlated significantly with their specific IgE antibody to AgE measured by RIA (r = 0.47; p less than 0.01) and with skin-test sensitivity to AgE (r = 0.44; p less than 0.05). It was concluded that CRIE is well suited for identification of important ragweed allergens without the previous need for laborious isolation procedures.
A crude extract of short ragweed pollen was obtained by extraction, centrifugation, dialysis and freeze-drying. Crossed immunoelectrophoresis, with a high-titer purified rabbit antibody fraction, revealed that the extract contained at least 52 antigens, of which 33 migrated toward the anode, 16 toward the cathode, and 3 toward both the anode and cathode. The electrophoretic precipitation pattern indicated a great deal of electrophoretic heterogeneity in several of the antigens. The overall distribution of apparent m.w. for 35 of the 52 antigens and the approximate m.w. for 19 identified antigens were determined by a combination of gel filtration and immunochemical analysis. Among antigens with m.w. weights less than or equal to 10,000, 8 of 10 were basic; on the other hand, all 5 identified antigens with m.w. above 40,000 were acidic. The apparent pI distribution for 30 antigens, and more precise pI values for 20 of 52 antigens, were determined by means of sucrose-gradient isoelectric focusing. The data obtained provide a reproducible reference system of considerable use in ragweed allergen chemistry and enable one to predict conditions for isolation of ragweed antigens.
We report a clinical study comparing the skin test reactivity to recombinant Aspergillus fumigatus allergen 1 (rAsp fI/a) in patients with atopic dermatitis and A. fumigatus sensitisation (n = 15), A. fumigatus-allergic patients with asthma (n = 10) and healthy control subjects (n = 10). All patients sensitised to A. fumigatus reacted at intradermal skin tests with commercial A. fumigatus extracts in contrast to the healthy subjects. Six out of 10 patients with well-characterised A. fumigatus allergic asthma were sensitised to rAsp fI/a as shown by a positive skin test. The patients with skin test reactivity to rAsp fI/a also showed rAsp f I/a-specific serum IgE as determined by ELISA. None of the patients with atopic dermatitis, healthy control subjects and 4 out of 10 A. fumigatus-allergic asthmatics reacted in intradermal tests to rAsp fI/a. Serologic investigations revealed that these subjects did not express detectable amounts of rAsp fI/a-specific IgE in agreement with the negative skin test results. Extended serologic investigations have not shown significant differences in rAsp fI/a-specific IgA, IgG4 and IgG1 serum levels between atopic dermatitis patients and healthy control subjects. The results suggest that sensitisation to A. fumigatus in patients with atopic dermatitis is not related to the major A. fumigatus allergen I in contrast to the high incidence of sensitisation to Asp fI/a occurring in allergic asthmatics.