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Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus
Nayak, A. P., B. J. Green, E. Janotka, J. M. Hettick, S. Friend, S. J. VESPER, D. Schmechel, AND D. H. Beezhold. Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus. CLINICAL AND VACCINE IMMUNOLOGY. American Society for Microbiology, Washington, DC, 18(9):1568-1576, (2011).
Approximately 9% of children have asthma. The medical costs of asthma are approximately $15 billion per year in the US alone and asthma results in about 2,000 deaths per year (Fisk et al. 2007). Lost school and work days run into the millions each year. The IOM’s expert committee (2004) concluded that exposure to moldy, damp indoor environments was associated with asthma. A subsequent review (Sahakian et al. 2008) of more recent publications also linked dampness to mold and asthma/asthma symptoms. The World Health organization has come to the same conclusion and suggest that exposure to molds should be “minimized” (WHO 2009). A meta-analysis of studies associating mold contamination with adverse health effects demonstrated that building dampness and mold were associated with approximately a 30 to 50% increase in a variety of respiratory and asthma-related health outcomes (Fisk et al. 2007). Therefore, it is critical that mold assessments are accurate and meaningful. US EPA researchers developed a DNA-based method of mold analysis called MSQPCR which is sensitive, specific and accurate. The US EPA in conjunction with HUD developed a simple, standard method of sampling homes for mold populations and created a scale called the ERMI to compare the mould burden in homes across the US (Vesper et al. 2007). In four epidemiological studies, higher ERMI values in homes were associated with increased risk of asthma in children. Remediating the water-damage and mould in asthmatics homes resulted in a statistically significant improvement in the child’s health and a reduction in the need for hospitalizations and emergency room visits (Kercsmar et al. 2006). Although these results are from a limited number of studies and have not been corroborated independently by other research groups, they are suggestive of the conclusions that mold problems are not always obvious. Furthermore, discovering hidden mold is possible using the ERMI analysis and subsequently correcting a mold problem may reduce asthma’s costs to the US by reducing hospitalizations and emergency room visits.
Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.