Identification of In Vivo Induced A. fumigatus Antigens

由烟曲霉菌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发病机制的认识不足，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)，我们在体内诱导抗原技术(IVIAT)中使用了识别HIV感染患者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来鉴定在人类IPA发病过程中诱导的烟曲霉菌抗原。在第一个特定目标中，我们将吸附从患者身上回收的配对血清：a)在诊断之前，以及b)从IPA中恢复后，对抗体外存在的烟曲霉菌抗原。然后，这些吸附的血清对将被用于平行筛选烟曲霉菌基因组DNA表达文库，以鉴定表达 免疫原性抗原。在IPA的发病过程中，可能会表达与恢复后血清中的抗体发生反应而与诊断前血清中的抗体不发生反应的抗原。配对的血清还将用于平行筛选cdna表达文库。在第二个特定目标中，将识别编码免疫原性抗原的基因。在第三个特定目标中，我们将纯化重组抗原，提高抗体，并使用它们来定位IPA小鼠肺内烟曲霉菌细胞的抗原。最后，在特定的目标4中，我们将比较针对体内诱导的抗原的血清抗体反应：a)IPA患者组和未感染的对照组，以及b)IPA诊断前和康复后的单个患者。我们推测，选定的体内诱导蛋白是烟曲霉的毒力因子，因此，可能代表治疗、疫苗或诊断靶点。这些假说将在未来的研究中得到验证，这将类似于目前的计划拨款中提出的表征活体诱导的白色念珠菌蛋白的研究。

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.