Identification of In Vivo Induced A. fumigatus Antigens

侵袭性肺曲霉病（IPA）由A。烟曲霉菌已成为免疫抑制患者死亡的主要原因。由于对IPA的发病机制了解不足，新的治疗、疫苗和诊断策略的开发受到限制。在与Nguyen博士的持续合作中（项目I），我们使用体内诱导抗原技术（IVIAT）来鉴定C。在HIV感染患者的口腔鹅口疮期间诱导的白色念珠菌蛋白，包括新的毒力因子。在这个项目中，我们将采用IVIAT来识别A。烟曲霉抗原在人类IPA发病过程中被诱导。在第一个具体目标中，我们将吸附从患者中回收的配对血清：a）诊断前，和B）从抗A的IPA中恢复后。烟曲霉抗原存在于体外。然后将吸附的血清对用于筛选A。烟曲霉基因组DNA表达文库，鉴定表达 免疫原性抗原。与恢复后血清中的抗体反应但与诊断前血清中的抗体不反应的抗原可能在IPA发病过程中表达。配对的血清也将用于平行筛选cDNA表达文库。在第二个具体目标中，将鉴定编码免疫原性抗原的基因。在第三个具体目标中，我们将纯化重组抗原，制备抗体并使用它们定位抗原到A。小鼠肺内的烟曲霉细胞与IPA。最后，在具体目标4中，我们将比较以下两组患者对体内诱导抗原的血清抗体应答：a）IPA患者组和未感染对照组，以及B）IPA诊断前和恢复后的个体患者。我们假设，选择在体内诱导的蛋白质是A。烟曲霉毒力因子，因此可能成为治疗、疫苗或诊断靶点。这些假设将在未来的研究中进行检验，这些研究将与目前计划资助中提出的描述体内诱导C。albicans蛋白。

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.