Identification of In Vivo Induced A. fumigatus Antigens

由烟曲霉引起的侵袭性肺曲霉病（IPA）已成为免疫抑制患者死亡的主要原因。由于对IPA发病机制了解不足，新的治疗、疫苗和诊断策略的发展受到限制。在与Nguyen博士（项目一）的持续合作中，我们使用体内诱导抗原技术（IVIAT）鉴定hiv感染患者口疮期间诱导的白色念珠菌蛋白，包括新的毒力因子。在本项目中，我们将采用IVIAT技术来鉴定在人类IPA发病过程中诱导的烟曲霉抗原。在第一个特定目标中，我们将吸附从患者身上恢复的配对血清：a)诊断前，b)从体外存在的烟曲霉抗原的IPA恢复后。这对吸附的血清随后将被用于平行筛选烟熏假单胞菌基因组DNA表达文库，鉴定表达的克隆

Invasive pulmonary aspergillosis (IPA) due to A. fumigatus has emerged as a leading cause of mortality among immunosuppressed patients. The development of new therapeutic, vaccine and diagnostic strategies is limited by poor understanding of the pathogenesis of IPA. In an ongoing collaboration with Dr. Nguyen (Project I), we used In Vivo Induced Antigen Technology (IVIAT) to identify C. albicans proteins that are induced during oral thrush in HIV-infected patients, including novel virulence factors. In this project, we will adapt IVIAT to identify A. fumigatus antigens that are induced during the pathogenesis of IPA in humans. In the first specific aim, we will adsorb paired sera recovered from a patient: a) before the diagnosis, and b) after recovery from IPA against A. fumigatus antigens present in vitro. The pairs of adsorbed sera will then be used to screen an A. fumigatus genomic DNA expression library in parallel, identifying clones expressing immunogenic antigens. Antigens reactive with antibodies in the post-recovery serum but not reactive with antibodies in the pre-diagnosis serum are likely to be expressed during the pathogenesis of IPA. The paired sera will also be used to screen a cDNA expression library in parallel. In the second specific aim, the genes encoding the immunogenic antigens will be identified. In the third specific aim, we will purify recombinant antigens, raise antibodies and use them to localize antigens to A. fumigatus cells within the lungs of mice with IPA. Finally, in specific aim 4, we will compare serum antibody responses against in vivo induced antigens among: a) groups of patients with IPA and uninfected controls, and b) individual patients before the diagnosis and after recovery from IPA. We hypothesize that selected in vivo induced proteins are A. fumigatus virulence factors and, as such, might represent therapeutic, vaccine or diagnostic targets. These hypotheses will be tested in future studies, which will be similar to those proposed in the present Program Grant to characterize in vivo induced C. albicans proteins.