Recombinant Aspergillus fumigatus Allergens: From the Nucleotide Sequences to Clinical Applications

Abstract

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.

Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies

“…serum IgE from A. fumigatus-sensitized individuals [9]. An intracellular Mn superoxide dismutase (Asp f 6) induces proliferation of peripheral blood mononuclear cells and elicits skin reactions in ABPA patients [10].…”

Overview of Aspergillus Allergens

“…However, the availability of the complete genome sequence allowed to verify all reported cDNA sequences encoding A. fumigatus allergens for consistency and for the presence of homologous and orthologous allergens within the fungal kingdom [11,22,23]. The detailed comparison between genome sequence and published cDNA sequences [23] revealed several Overview of Aspergillus Allergens inconsistencies: (i) Asp f 15 [24] and Asp f 13 [25] are identical; (ii) Asp f 16 [26] does not appear to be encoded by the A. fumigatus AF293 genome but shows a high degree of homology with Asp f 9 [27], which is present in the genome. Therefore it can be concluded that the Asp f 9 cDNA sequence is correct, and that the published Asp f 16 peptide sequence results from frameshift, other sequencing errors, or derives from a different A. fumigatus strain; (iii) Asp f 17, originally described as a cDNA encoding a 191 amino acid protein [24], is incomplete and the complete gene encodes a protein of 284 amino acids; and (iv) the postulated sequence of the Asp f 56 kDa allergen not included in the official allergen database, which was derived from a short peptide obtained from a biochemically purified protein [28] is not predicted to be encoded in any of the sequenced Aspergillus genomes.…”

“…The whole repertoire of A. fumigatus allergens included in the official allergen list reported in Table 2 have been produced as highly pure recombinant allergens and evaluated for their IgE-binding capacity in vitro. The majority of these proteins have also been tested for their ability to elicit positive skin tests in vivo [27,[29][30][31][32].…”

“…As shown in Table 2, rAsp f 1, 3, 4-11, 15, 17, 27-29, and 34 have been investigated in skin test studies. The experience accumulated performing skin tests and serology with different recombinant A. fumigatus allergens involving more than 600 individuals suffering from various A. fumigatus-related diseases and over 100 healthy controls allows to draw the following firm conclusions: (i) only individuals with detectable serum IgE levels against a tested recombinant allergen showed positive skin test reactions with the specific allergen [44]; (ii) the overall positive skin test response of individuals sensitised to A. fumigatus extracts to recombinant allergens are highly variable from allergen to allergen ranging from around 70% for the major allergen rAsp f 1 [27] to a few percentage for minor allergens like Asp f 8 or Asp f 10 [24]; (iii) no reactivity to any of the recombinant allergens could be detected in any of the more than 100 healthy controls tested; (iv) rAsp f 4 and rAsp f 6 are predominantly recognised by patients suffering from ABPA [32, …”

Immune response to n-terminal and c-terminal deletion mutants of Aspergillus fumigatus major allergen ASP F 3

“…The involvement of Af-specific Th1 cytokines in allergic response raise the possibility of uncovering effective therapeutic modalities that would promote Th1 immune response and /or T-cell tolerance through regulatory T cells (CD4+ CD25+) in Af sensitized patients. In recent years, major Af allergens have been identified and purified using molecular cloning and expression protocol (3)(4)(5)(6). Asp f 3 has been recognized as a major allergen of Af, which elicited immediate wheal and flare skin reactions and strong IgE binding with a majority of ABPA patients' sera (1,4,7).…”