Use of a Synthetic Peptide Epitope of Asp f 1, a Major Allergen or Antigen ofAspergillus fumigatus, for Improved Immunodiagnosis of Allergic Bronchopulmonary Aspergillosis

Abstract

Allergic bronchopulmonary aspergillosis (ABPA) is an immunologically complex allergic disorder caused by the fungal pathogen Aspergillus fumigatus. Elevated levels of total immunoglobulin E (IgE), specific IgE, and IgG antibodies in sera are important immunodiagnostic criteria for ABPA. International reference standards or standardized immunodiagnostic assays are not available due to a lack of well-defined diagnostic antigens. The present study was carried out to identify and evaluate the immunodiagnostic relevance of synthetic epitopic peptides of Asp f 1, a major allergen, antigen, or cytotoxin of A. fumigatus. Five overlapping peptides were synthesized from the N terminus of Asp f 1, one of the potential immunodominant regions predicted by algorithmic programs. The 11-amino-acid synthetic peptide (P1) significantly inhibited both IgG binding (89.10% ؎ 4.45%) and IgE binding (77.32% ؎ 3.38%) of the standardized diagnostic antigen (SDA) (a well-defined pool of diagnostically relevant allergens and antigens of A. fumigatus). With a panel of sera of ABPA patients, allergic patients with skin test negativity to A. fumigatus, and healthy individuals, P1 showed a higher diagnostic efficiency than SDA (specific IgG, 100%; specific IgE, 98.3%). The diagnostic efficiency of P1 could be attributed to the presence of homologous epitopes in various immunodominant allergens or antigens of A. fumigatus. The ability of P1 to induce histamine release from sensitized mast cells and a Th2 type of cytokine profile in peripheral blood mononuclear cells of ABPA patients suggests its potential for use in intradermal testing. P1 could be further explored for development of a standardized, specific, and sensitive immunodiagnostic test for aspergillosis.

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

“…Images were generated by the MOLMOL program [30] and subsequently rendered with MegaPov. (C) Sequence alignments of the recombinant proteins Aspf1, Aspf1D (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), and a-sarcin [11,16,31]. The deleted portion and the two substituting Gly in Aspf1D (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) are marked in bold characters.…”

“…(C) Sequence alignments of the recombinant proteins Aspf1, Aspf1D (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), and a-sarcin [11,16,31]. The deleted portion and the two substituting Gly in Aspf1D (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) are marked in bold characters. The recombinant Aspf1 and Aspf1D (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) have an extra Val residue at the seco...…”

“…The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1D (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) have been produced as recombinant proteins; the deleted region corresponds to an exposed b-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy.…”

“…Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1D (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted b-hairpin is involved in ribosome recognition and is a significant allergenic region.…”

Aspergilli—aflatoxins: health threat

“…This scenario poses a major global scientific challenge for understanding & Usha P. Sarma pusarma@gmail.com the aflatoxin biosynthesis in order to develop preventive and management strategies for the toxigenic fungus and the toxin. Similarly specific, sensitive and affordable commercial diagnostic reagents are a necessity today for prevention and management of Aspergillus related diseases in humans and animals [4,5]. Above challenges are being countered with the current knowledge of genomic and proteomic aspects of Aspergilli.…”