Cloning and expression of Aspergillus fumigatus allergen Asp f 16 mediating both humoral and cell-mediated immunity in allergic bronchopulmonary aspergillosis (ABPA)

BACKGROUND: Aspergillus fumigatus, a ubiquitous fungus, is responsible for a number of lung disorders in atopic and non-atopic individuals. Standardized, pure, and relevant allergens are desirable for reliable immunodiagnosis of the disease and to understand the structural and functional properties of these allergens and the role they play in causing ABPA. OBJECTIVE: Molecular cloning and characterization of a relevant allergen from A. fumigatus cDNA library. MATERIALS AND METHODS: A cDNA library was constructed from 96 h old mycelium of A. fumigatus using lambda ZAP expression vector. A novel gene encoding an A. fumigatus allergen was identified by screening the library with sera from ABPA patients. The gene was cloned and the allergen over-expressed in Escherichia coli. This recombinant allergen, Asp f 16, was evaluated in ELISA and Western blots using sera from patients and normal subjects and peripheral blood mononuclear cells (PBMC) for antigen-induced stimulation. RESULTS: Seventy percent of the patients with ABPA demonstrated high levels of serum IgE antibodies to Asp f 16, a 43-kDa protein, whereas patients with allergic asthma, Aspergillus skin test-positive asthmatics without clinical evidence of ABPA, and normal controls failed to show Asp f 16-specific IgE binding by ELISA. The deduced amino acid sequences of Asp f 16 showed extensive sequence homology to 30.6-kDa Asp f 9 at the N-terminal region of the protein. PBMC from the majority of patients with ABPA exhibited significant proliferation with the recombinant Asp f 16 allergen. CONCLUSION: Specific humoral and cell-mediated immune responses of Af-sensitized patients against Asp f 16 suggest its usefulness in the immunodiagnosis of hypersensitivity diseases due to Af and understanding the pathophysiology of ABPA.     

Differential IgE recognition of recombinant Aspergillus fumigatus allergens by cystic fibrosis patients with allergic bronchopulmonary aspergillosis or Aspergillus allergy

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.     

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.     

Molecular and immunological characterization of Asp f 34, a novel major cell wall allergen of Aspergillus fumigatus

Background:  Although fungal spores have been recognized as triggers of respiratory allergy and asthma, only two allergenic fungal cell wall components have so far been described.
Methods:  Eighty-one sequences derived from an Aspergillus fumigatus cDNA library encoding putative allergens were examined for the presence of cell wall components. A new allergen (Asp f 34) was evaluated by Western blots, enzyme-linked immunosorbent assay (ELISA), peripheral blood mononuclear cell (PBMC) proliferation assays, and skin prick test (SPT).
Results:  The cDNA encoding Asp f 34 contained an open reading frame predicting a protein of 185 amino acids with a molecular weight of 19.38 kDa, showing sequence homology to phiA, an essential protein for the formation of conidia in the genus Aspergillus . The recombinant Asp f 34 was binding IgE from sensitized individuals in Western blots. An ELISA survey showed that 94% of the ABPA and 46% of the A. fumigatus -sensitized individuals tested had Asp f 34-specific serum IgE. Asp f 34 induced allergen-specific proliferation exclusively of PBMCs from patients sensitized to the allergen. Eight patients with anti-Asp f 34 serum IgE tested reacted positively in SPT, whereas four A. fumigatus -sensitized individuals without Asp f 34-specific IgE and eight healthy controls scored negatively.
Conclusions:  A cell wall protein of the phialides of A. fumigatus was identified as a major allergen. Asp f 34 belongs to the Aspergillus -specific proteins of the phiA family and has relevant potential for a specific diagnosis of Aspergillus sensitization.     

Hyperreactivity of mediator-releasing cells from patients with allergic bronchopulmonary aspergillosis as evidenced by basophil histamine release

Histamine release was performed on 28 patients with allergic bronchopulmonary aspergillosis (ABPA) in various stages and on 14 patients with mold-sensitive asthma, by using Aspergillus antigens, other mold antigens, and anti-IgE. Total serum IgE, IgE and IgG antibodies to Aspergillus fumigatus (Af), and end point cutaneous titration for Af were determined for each patient. There was greater histamine release to Aspergillus mix (p = 0.0000) and anti-IgE (p = 0.047) in patients with ABPA than in patients with mold-sensitive asthma. Patients with stages IV and V ABPA had greater histamine release to Aspergillus mix than patients with stage I, II, or III ABPA (p = 0.0014). There was greater histamine release to other molds in ABPA patients than in mold-sensitive asthmatics. There was no correlation between histamine release to Aspergillus mix and total serum IgE, IgE or IgG antibodies to Af, ratio of IgE and IgG antibodies to Af to total IgE, or cutaneous end point titration for Af in ABPA patients or mold-sensitive asthmatics. Peripheral basophils from ABPA patients demonstrated marked hyperreactivity as evidenced by histamine release to Aspergillus antigens, other molds, and anti-IgE. This hyperreactivity is the first cellular difference recognized in ABPA patients when compared to mold-sensitive asthma patients. If this hyperreactivity is manifested by mediator release from other cells such as pulmonary mast cells, which would be in close contact with growing Af, a possible mechanism for pulmonary response in ABPA would be suggested.     

Anti-Candida albicans IgE and IgG subclasses in sera of patients with allergic bronchopulmonary aspergillosis (ABPA)

We performed immunoblotting experiments to determine specific IgE and IgG subclass responses to Candida albicans antigens in allergic bronchopulmonary aspergillosis (ABPA) patients. This is a first report describing C. albicans antigens recognized by serum IgE and IgG subclasses of ABPA patients sensitized to that yeast. Among the various antigens reacting with serum IgE, a 43-kDa component was recognized by all seven patients and can be considered a major antigen of C. albicans for this particular group of patients. By comparison, only 20% of a group of asthmatic atopics (25 patients) and 10% of a group of normal controls (10 subjects) were 43-kDa positive. Multiple banding patterns, revealing no major antigen, were observed for all four IgG subclasses except for IgG1 in one case. In particular, the 43-kDa component was not always recognized by all the patients. Furthermore, oral or inhaled steroid treatment appears to have no impact on the specific IgE immunopatterns obtained. Using immunoelectron-microscopy, we localized IgE-binding primarily in the mannoprotein-containing layers of the C. albicans cell wall. In conclusion, C. albicans-IgE and IgG subclasses may participate in the physiopathology of ABPA by exacerbating pulmonary infiltrates (IgE) and inducing eosinophil-mediated inflammatory reaction (IgG1, IgG3).