Target Discovery and Validation for Immunodiagnosis of Invasive Aspergillosis
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Authors
Chaves, Sindy J.
Issue Date
2012
Type
Dissertation
Language
Keywords
Antibodies , Immunodiagnosis , immunogenic peptides , InMAD , Invasive Aspergillosis , mass spectroscopy
Alternative Title
Abstract
nvasive aspergillosis (IA) is a serious opportunistic infection caused mainly by Aspergillus fumigatus. Symptoms of IA are often non-specific, making the disease difficult to diagnose. The goal of this study is to develop a sensitive, noninvasive and inexpensive immunoassay for point-of-care diagnosis of IA. A critical step in development of an immunodiagnosis strategy that detects antigen is to identify antigens that are shed into body fluids during infection in concentrations sufficient for detection. For this purpose, we developed a novel strategy termed In vivo Microbial Antigen Discovery (InMAD). InMAD is based on the hypothesis that serum or urine from animal models of IA contain precisely those antigens that would be targets for immunoassay. In the InMAD technique, immunosuppressed mice and guinea pigs were infected via the pulmonary route with A. fumigatus spores. Serum and urine were collected and filtered to remove whole fungal cells but leave behind soluble antigens released during infection. The filtered samples were used to immunize naïve mice, which see the fungal antigens as foreign and make antibodies. Serum was collected from the immunized mice and used to identify antigens through use of immunoblots prepared from 1D and 2D gels containing fungal whole cell lysates. All samples from the different animal models of IA generated antibody responses to A. fumigatus antigens when evaluated by 1D and 2D immunoblots. Sera from mice producing antibody responses were pooled and used to probe 2D blots of whole cell lysates. The results produced a high-resolution profile of many A. fumigatus proteins that were shed into serum and urine during infection. Mass ii spectroscopy analysis of reactive proteins identified fifteen candidate antigens that may be used for immunodiagnosis of IA. Significantly, those candidate antigens were identified in both serum and urine, and at least four of the antigens were found in both mouse and guinea pig models of IA. Bioinformatics analyses were done to assess the uniqueness of each protein. In addition, five of those target antigens were selected and polyclonal antibodies (pAbs) were produced to immunogenic sequences of those proteins. Immunoblots of serum and urine from A. fumigatus-infected guinea pigs that were probed with the pAbs showed reactive spots that corresponded to the proteins to which those antibodies were made. Furthermore, immunoblots of serum from humans with a high probability of IA were probed with the pAbs, and those reactive spots were also found to correspond to the proteins to which those antibodies should interact with. These results indicate that several A. fumigatus proteins are shed into serum and urine during infection and that these antigens can be identified by the InMAD technique. Importantly, potential immunoassay targets identified from study of mouse and guinea pig models of IA were also detected when pAbs to immunogenic regions of those targets were used against an A. fumigatus infected sample of guinea pig serum and urine. These results suggest that it may be possible to construct an immunoassay for detection of A. fumigatus proteins that will be useful in diagnosis of IA.
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In Copyright(All Rights Reserved)