Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma

Abstract

IntroductionSevere equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA.MethodsAiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.Results and discussionFor 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma

“…However, this polarization was not very clear regarding the total serum Ig isotype contents and was not reflected in Aspergillus- binding Ig in MEA. Additionally, the previously reported serum IgG3/5 bias in SEA for A. fumigatus antigen binding on immunoblots ( 24 , 70 ) could not be corroborated in this study, which included a larger and different cohort of horses. The previous study on horses with SEA compared to HE analyzed serum Ig binding to A. fumigatus on immunoblots ( 24 ) and different methods likely contribute to the different results compared to ELISA analyses here.…”

“…A. fumigatus strain CBS 144.89 (CEA10), provided by Dr. Olaf Kniemeyer, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany, was grown on semi-solid growth medium (1% mycological peptone, Thermo Fisher Scientific, Waltham, MA, USA) and potassium phosphate buffer, pH 7.0 [3.4 mM KH 2 PO 4 , 5.75 mM K 2 HPO 4 , 0.06% thiamine, 2 mM MgCl 2 , 100 µg/mL chloramphenicol, gentamicin, chlortetracycline, and 20% (w/v) Kolliphor ® P 407, Sigma-Aldrich] at 37°C for 1–2 days, as previously described ( 24 ). A. fumigatus mycelium with spores was harvested, washed, and lysed by sonication in buffer {7 M urea, 2 M thiourea, and 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), Carl Roth, Karlsruhe, Germany}, and cleared by centrifugation and filtration, as previously described ( 24 ). The protein concentration was determined with the Bradford method using ROTI ® Quant (Carl Roth) and albumin standard (Thermo Fisher), accounting for the buffer in the standard and blanks of the assay ( 49 ).…”

“…The allergens Asp f 1, Asp f 7, and Asp f 8 of A. fumigatus were recombinantly expressed in Escherichia coli ( E. coli ) and purified as previously described ( Table 1 ) ( 21 , 24 , 50 – 52 ).…”

“…Additional antigens were prepared as previously described ( Table 1 ; Supplementary Table 1 ) ( 24 ). Briefly, coding sequences of the antigens of interest from strain A. fumigatus CBS 144.89 were cloned into the expression vector pET28a (+) (Sigma-Aldrich, St. Louis, MO, USA) propagated in chemically competent E. coli strain Rosetta pLysS (Sigma-Aldrich), cultured on lysogeny broth (LB)-agar plates containing 15 µg/mL kanamycin and 34 µg/mL chloramphenicol (Euroclone, Pero, Italy), grown overnight at 37°C, followed by a liquid sub-culture in LB medium containing the same antibiotics to prepare cryo-stocks, which were stored in 60% glycerol in LB medium at −80°C.…”

“…A. fumigatus protein antigens considered in EA have also been identified as allergens in human diseases [Asp f 1, Asp f 7, Asp f 8, and dipeptidyl-peptidase 5 (DPPV)], but some of these (Asp f 7 and Asp f 8) have been repeatedly suggested as antigens in analyses of EA ( 17 – 23 ). Additional A. fumigatus protein antigens that have not been described as allergens were identified by a bottom-up immunoproteomics approach analyzing binding of serum IgG from SEA ( 24 ). It is unclear if A. fumigatus allergens defined by human IgE reactivity are also the dominant immunogens for horses, or if there is a general dominance of single antigens in EA as implied by equine IgE binding profiles on several available allergen preparations ( 19 , 22 ).…”