Recombinant Allergens from A. fumigatus:
A large number of recombinant allergens from pathogenic strains of A. fumigatus have been cloned, produced as recombinant proteins, characterized and partly evaluated for their diagnostic value in ELISA and skin tests (Crameri, 1998a; Kurup et al., 2000). The recent listing of IUIS-WHO Allergen Nomenclature Committee includes 22 allergens from Aspergillus and Penicillium species (Table 1). However, not all of these allergens have been evaluated for their role in allergy or in their ability to bind to IgE in the sera of sufficient number of patients. Only those allergens evaluated in IgE binding assays which have shown IgE-reactivity with a minimum number of patients' sera (As recommend by the Allergen Nomenclature Committee; King et al., 1995) are discussed. The molecular structures cover a wide range of functional proteins including toxins, enzymes, heat shock proteins and several proteins lacking homology to the known proteins deposited in the data protein banks.
Asp f 1 - Ribotoxin:
Asp f 1, a ribotoxin, which inhibits protein translation, is a major 18kDa allergen from A. fumigatus. This allergen was initially purified from the mycelium of A. fumigatus extracted with borate buffered saline and used to produce mouse monoclonal antibodies. Pure native allergen was obtained by affinity chromatography purification using a monoclonal antibody, mAb 4A6. The amino acid sequence comparison demonstrated 99% homology with mitogillin, a cytotoxin produced by A. restrictus (Arruda et al., 1990). This allergen showed skin test reactivity and binding to serum IgE of patients with ABPA. The cDNA encoding Asp f 1 was cloned and shown to spans 686 base pair in length containing an open reading frame predicting a protein with a calculated MW of 16.9 kDa (Moser et al., 1992). The allergen was expressed in Escherichia coli as a fusion protein with six Histidine tag (Moser et al., 1992) and purified using Ni+-chelate affinity chromatography. Arruda et al. also cloned the gene encoding the Asp f 1 allergen, a protein of 149 amino acid residue (Arruda et al., 1990). The entire 678 base pair DNA from A. fumigatus conidia include 81 by leader sequence, preceding the N-terminal alanine codon, a 52 by intron, and a 447 by open reading frame.
Asp f 1 was present in high concentrations in the culture filtrates of A. fumigatus compared to the spores and hyphae suggesting that the higher level of specific antibody seen in sera of allergic patients may be due to the secretion of Asp f 1 by the germinating spores present in the bronchus and airways. This allergen was not detected in other Aspergillus species with the exception of A. restrictus (Lamy and Davies, 1991a) and different clinical isolates of A. fumigatus (Kao et al., in press; Lamy et al., 1991b; Moser et al., 1993). The recombinant Asp f 1 was found to be toxic to EBV-transformed or PHA-stimulated PBMCs (Moser et al., 1992).
Asp f 1 studied by Western blot, ELISA and radioimmunoassays demonstrated specific reactivity with sera from patients with ABPA or are sensitized with A. fumigatus, but not with sera from normal subjects (Arruda et al., 1990; Moser et al., 1992). Recombinant Asp f 1 showed skin test reactivity in eight out of nine patients with ABPA and in five out of ten A. fumigatus-sensitized allergic asthmatics, while all seven normals and six non-Aspergillus allergic patients showed no reactivity (Moser et al., 1992; Moser et al., 1994a). The reactivity with Asp f 1 was shown to be specific as none of the atopic dermatitis or non-Aspergillus allergic asthma patients reacted to this allergen (Disch et al., 1995). This allergen was also found to be specific in A. fumigatus-sensitized patients suffering from cystic fibrosis with or without ABPA (Nikolaizik et al., 1995). All the six ABPA patients from a total of 44 cystic fibrosis showed positive skin prick test and specific IgE in ELISA against rAsp f 1 (Nikolaizik et al., 1996). In a recent evaluation of 24 allergic asthmatics suffering from ABPA, 68-83% of ABPA patients showed strong IgE response to rAsp f 1 in ELISA (Kurup et al., 2000). Thus, Asp f I is a major allergen for skin test reactivity and in vitro IgE binding assays (Table 2). Asp f 1 was the first allergen tested for automated IgE-binding assay in the Pharmacia CAP System and demonstrated the feasibility of using recombinant allergens in automated serological diagnosis of allergic diseases (Crameri et al., 1996a; Crameri et al., 1996b).
The IgE binding to linear region of the molecules demonstrated 13 epitopes. When these peptides were synthesized and evaluated by ELISA several of them reacted directly with the serum IgE from ABPA patients. A number of these epitopes containing synthetic peptides inhibited the IgE binding of Asp f 1 in an ELISA inhibition assay (Kurup et al., 1998). Several T-cell stimulatory epitopes were also identified from Asp f 1.
Asp f2:Aspergillus fumigatus major allergen Asp f 2 is a 310 amino acid protein having fibrinogen binding property (Banerjee et al., 1999). This gene has two introns of 83 and 52 base pairs each and four potential glycosylation sites. cDNA has a 126 base pair long signal sequence and an 804 base pair long open frame reading. There was strong sequence homology shown with Asp ndl, a fibrinogen binding protein from A. nidulans and with pH regulatory protein from Candida albicans (Banerjee et al., 1999). No other proteins in the data bank showed any homology with Asp f 2. The native protein purified using monoclonal antibody affinity chromatography demonstrated a size of 37kDa, while the E. coli expressed Asp f 2 showed a size of 32-35kDa. However, when the protein was expressed in the eukaryotic host Pichia pastoris it showed a size of 70-75kDa. On deglycosylation the molecular size returned to a size of 37-42kDa, comparable to the E. coli expressed protein and native Asp f 2 (Tang et al., 2000). The secretion of this protein by different strains of A. fumigatus varied considerably and the peak secretion was noted on day seven in the aerated cultures (Banerjee et al., 1997). The Pichia expressed proteins showed more reactivity with IgG antibody in the sera of ABPA patients, asthmatics, and normals, while the E. coli expressed proteins and native allergens bound to both IgE and IgG. Peptides expressed by deletion mutants differed in their binding activity with IgE and IgG (Banerjee et al., 1999; Tang et al., 2000). IgE binding epitope analysis demonstrated nine linear regions spanning the whole sequence and several of them may be found to be at least partly conformational as indicated by competitive inhibition of IgE binding of Asp f 2 (Banerjee et al., 1999; Tang et al., 2000). The selectively deleted regions containing epitopes also confirmed these results (Tang et al., 2000). Similarly some of the deletion mutants failed to bind to IgE or inhibited the binding of Asp f 2. The reactivity of Asp f 2 with patient and control sera is shown in Table 2.
Asp f 3 - Putative peroxisomal protein:
A putative peroxisomal membrane protein of A. fumigatus, Asp f 3, is encoded by a 616 by long cDNA (Hemmann et al., 1997). The open reading frame encompasses 507 by and encodes a protein of 168 amino acids with a calculated molecular mass of 18.5 kDa. This allergen showed homology with two peroxisomal proteins of Candida boidinii, IgE reactivity with sera from Aspergillus allergic patients in Western blot and in ELISA and significant cross reactivity with two Candida peroxisomal proteins (Hemmann et al., 1997). However, crossinhibition studies demonstrated the presence of Asp f 3-specific binding epitopes for IgE from serum of A. fumigatus-sensitized allergic asthmatics in addition to the shared epitopes between the two Candida proteins and Asp f 3 (Hemmann et al., 1997). This allergen was not found to be cytotoxic to CTLL even up to a concentration of 1 mg. Asp f 3 elicited positive skin test reactions in patients and bound to IgE from A. fumigatus-sensitized allergic asthmatics and CF patients with ABPA (Crameri, 1998; Hemmann et al., 1998a; Hemmann et al., 1998b). A recent study suggests that this major allergen will be of value in the differential diagnosis of ABPA (Table 2) (Kurup et al., 2000).
Asp f 4 - Allergen with unknown biochemical function:
Asp f 4 a protein of 286aa with a predicted MW of 30kDa was isolated, like the majority of the A. fumigatus allergens, from a cDNA library displayed on the surface of filamentous phage (Crameri and Suter, 1993; Crameri et al., 1994; Crameri et al., 1998b; Crameri, 1999a; Hemmann et al., 1998a; Hemmann et al., 1998b). The cDNA coding for the mature proteins was subcloned into a high level expression vector and produces as N-terminal hexahistidine-tagged recombinant protein in E. coli (Crameri et al., 1998b; Hemmann et al., 1998b). Asp f 4 is an intracellular protein with no consensus sites for N-glycosylation without sequence homology with any of the known proteins in the data bank. This allergen also showed strong reactivity with IgE from the sera of both allergic asthmatics and cystic fibrosis patients suffering from ABPA (Crameri et al., 1998b; Hemmann et al., 1998b). Recently it has been shown that rAsp f 4 is able to elicit strong skin test reactions in patients suffering from ABPA but not in A. fumigatus-sensitized asthmatics suggesting that the allergen could be used as a specific marker for the diagnosis of ABPA (Crameri, 1999a, Hemmann et al., 1999). The results of the IgE binding assays using this allergen are summarized in Table 2.
Asp f 5 - Metalloprotease:
A. fumigatus secretes a metalloprotease (Jaton-Ogay et al., 1992; Jaton-Ogay et al., 1994; Moser et al., 1994) and an aspartic protease (Reichard et al., 1995), claimed to be allergens (Robinson et al., 1990) and virulence factors of the fungus (Monod et al., 1991). The mature protein for the Asp f 5 allergen spans 388aa with a predicted MW of 42.06kDa (Crameri, 1998a). The allergen was recognized by 26 of the 35 sera from allergic asthmatics without ABPA and by 50 of the 54 sera from patients with ABPA. Therefore, Asp f 5 has to be classified as a major allergen according to the allergen nomenclature rules (King et al., 1995). Asp f 5 has been shown to be able to elicit specific type 1 skin reaction in A. fumigatus-sensitized individuals and showed binding to IgE in Western blot analysis (Menz and Crameri, unpublished).
Asp f 6 - Manganese superoxide dismutase (MnSOD):
Asp f 6 is a manganese superoxide dismutase having a molecular
mass of 23 kDa spanning 207aa and was originally cloned from a
cDNA library displayed on the surface of the filamentous phage M13
(Crameri et al., 1996d). The library was enriched for phage
displaying gene product able to bind to human serum IgE with solid
phase-immobilized IgE from patients allergic to A.
fumigatus (Crameri et al., 1994; Crameri, 1998a). The
clone encoding MnSOD spans 751 base pairs in length with an open
reading frame of 630bp predicting a protein of 210 amino
acids. The gene product showed strong homology to MnSOD of human,
bovine, fruit fly, gum tree, yeast, and E. coli enzymes
(Mayer et al., 1997).
Asp f 6 shows 51% identity and 67.2% homology to the corresponding human enzyme. Both, the recombinant A. fumigatus and the human enzyme were purified by Ni+ chealate affinity chromatography, refolded in vitro and shown to exhibit the expected enzymatic activity (Crameri et al., 1996d; Mayer et al., 1997). These two recombinant proteins bound to serum IgE from patients allergic to A. fumigatus. On skin testing, these patients showed immediate skin test reactivity to both MnSODs. The binding of serum IgE from patients allergic to A. fumigatus to solid-phase-bound A. fumigatus MnSOD was inhibited by recombinant human MnSOD indicating the presence of shared B-cell epitopes (Crameri et al., 1996d). Both recombinant human and A. fumigatus MnSOD also induced proliferation of peripheral blood mononuclear cells of A. fumigatus allergic patients who had circulating IgE antibody and skin test reactivity to MnSOD. The immune response, both humoral and cellular, implies the presence of autoreactivity to human MnSOD in patients allergic to A. fumigatus (Appenzeller et al., 1999; Crameri et al., 1996d). rAsp f 6 has been postulated to be a specific marker for the diagnosis of ABPA based on both, IgE-binding studies (Crameri et al., 1998b; Crameri, 1999a; Hemmann et al., 1998b). Recently several MnSODs have been reported from other fungi and other sources such as latex (Wagner et al., 2000). It is not known whether T-cell and B-cell epitopes are shared by these groups of allergens. The reactivity of this allergen to patient sera is shown in Table 2.
rAsp f 8 - Acidic P2 ribosomal protein:
Acidic P2 ribosomal protein has been described as allergen cloned from Alternaria alternata and Cladosporium herbarum has also been isolated as IgE binding protein from A. fumigatus cDNA library displayed on phage surface as well (Crameri, 1999a). The allergen, formally termed Asp f 8 has a molecular weight of 11.1kDa and shares >62% sequence identity and >84% sequence homology to corresponding eukaryotic P2 proteins, including human P2 protein (Mayer et al., 1999). Both A. fumigatus and human P2 proteins were recognized by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited strong immediate type skin reactions in these individuals. The human protein was able to inhibit IgE binding to solid-phase coated A. fumigatus P2 protein in ELISA and induce proliferative responses in peripheral blood mononuclear cells of A. fumigatus-allergic subjects sensitized to the fungal P2 protein. As in the case of MnSOD, these available informations provide strong evidence for humoral and cell-mediated autoreactivity to this antigen in patients suffering from chronic A. fumigatus allergy (Mayer et al., 1999).
Asp f 9:
Asp f 9 is a 33.7kDa allergen with 302 amino acid residues. The sequence shows a low homology with the Ferrioxamin B permease gene of Saccharomyces cerevisiae. Serologic studies showed that the majority of the sera from ABPA patients tested (48 out of 54) had detectable levels of rAsp f 9-specific IgE antibody. However, I I out of 35 sera from allergic asthmatics without ABPA sensitized to A. fumigatus also showed significant amounts or rAsp f 9-specific IgE indicating that Asp f 9 is a minor allergen for this subclass of patients. According to the definition rAsp f 9 has to be regarded as a major A. fumigatus allergen since 66% of the 89 sera tested demonstrated IgE antibody to rAsp f 9 by ELISA.
Asp f 10 - Aspartic protease:
The mature aspartic protease of A. fumigatus contains 325aa with a predicted MW of 34.4kDa (Reichard et al., 1995). The allergen is recognized by only 16 of 89 allergic aspergillosis sera tested and hence, Asp f 10 is to be considered as a minor allergen of A. fumigatus (Crameri, 1998a). However, this protease has been demonstrated in the fungal elements and tissue infiltrates of fatal cases of invasive aspergillosis suggesting the production and secretion of protease in the lung tissue of infected patients (Reichard et al., 1995).
Asp f 11 - Cyclophilin (peptidyl-prolyl cis-trans isomerase):
Cyclophilin was first isolated in 1984 from bovine thymus (Handschumacher et al., 1984) and described as an enzyme playing a critical role in protein folding (Fischer et al., 1989; Takahashi et al., 1989). Cyclophilin was isolated as an IgE-binding protein from a Malassezia furfur cDNA library displayed on phage surface (Lindborg et al., 1999). The allergen, formally designated Mal f 6 shows 69% sequence identity to A. fumigatus cyclophilin (Asp f 11), an allergen previously cloned from an A. fumigatus phage surface display library (Crameri, 1998a). The A. fumigatus allergen was able to bind IgE from 50% of 60 sera from individuals sensitized to the fungus and hence, should be considered a relevant allergen associated with ABPA (Hemmann, 1998c).
Asp f 12 - Heat Shock Protein of Aspergillus:
A number of heat shock proteins have been reported from a large number of unrelated species (Kumar et al., 1993). We have identified a clone from a cDNA library of A. fumigatus reacting with IgE from ABPA patients. The protein was expressed as a fusion protein having a molecular size of 65kDa. This protein showed strong reactivity with IgE antibody in the sera of ABPA patients, but failed to react with normal sera. The truncated protein showed a homology with HSP 90 from a number of species including Candida albicans, Saccharomyces, Trypanasoma, house fly, mouse, human, and E. coli. Because of the extraordinary conservation of the HSPs and their potential immunogenicity, their role in infection and in autoimmunity are emphasized, particularly the stress response during inflammation. This allergen reacted with both IgG and IgE antibodies in allergic patients, although IgE antibody response against Asp f 12 is also detected in normal controls. Strong T-cell response has also been shown by this allergen in ABPA, but not with control subjects.
Asp f 16 - Allergen with unknown biological function:
Recently a 43kDa protein of A. fumigatus was expressed from a gene cloned in E. coli (Banerjee et al., in press). This protein Asp f 16 is a new allergen with no similarity to any of the proteins in the data bank. However, the sequence of this gene as well as the expressed protein showed strong homology with another allergen from Aspergillus, Asp f 9, and a lower degree of homology with Saccharomyces cerivisiae membrane protein (Crameri, 1998). Asp f 16 reacted with IgE from ABPA patient sera both by immunoblot and by ELISA. This gene is encoded by a cDNA spanning 1546bp with an open frame reading of 1281bp encoding a protein of 427 amino acids. There is an 18 amino acid long signal peptide. This allergen is rich in threonin, serine, and alanine and has a potential N-glycosylation site. Asp f 16 showed 100% specificity and 70% sensitivity in their IgE binding with sera from ABPA patients. There was no reactivity of this allergen with normal subjects. Strong PBMC stimulation was seen in ABPA patient with comparatively weaker response in allergic asthma patients, but no stimulation was demonstrable in the normal controls.
Other Allergens:
Several additional A. fumigatus allergens have been reported and are shown in Table 1. None of these proteins have been evaluated convincingly to warrant their inclusion as major allergens. However, several of these recombinant antigens may be of value in the diagnosis of ABPA and related diseases or may probably participate in the pathogenesis of allergic aspergillosis. For understanding their roles in immunological, histological, and physiological responses in human disease further studies are needed including investigations of animal models of allergy (Kurup et al., 2000).