Immunogenicity of recombinant chaperone-complexed antigens

我们将研究抗原/热休克蛋白复合物的免疫原性，这些抗原/热休克蛋白复合物要么被抗原呈递细胞（APC）表达（作为内源性复合物），要么被APC吸收作为外源性复合物，加工并呈递到淋巴细胞。将改进重组hsp73融合抗原复合物的生产、纯化和表征方法。将研究处理外源性或内源性hsp/抗原复合物以将MHC i类限制性表位呈递给细胞毒性T淋巴细胞（CTL）的细胞生物学。主要重点将放在外源性或内源性热休克蛋白/抗原复合物的细胞内运输、加工发生的亚细胞位点、呈递能力MHC I类分子产生的位点和动力学以及所涉及的蛋白水解系统上。将尝试通过在相关表位的两侧放置组织蛋白酶敏感的切割位点来促进热休克蛋白结合的重组融合抗原的蛋白水解加工。这些体外研究将辅以体内研究，阐明含有热休克蛋白/抗原复合物的基于DNA或蛋白质的疫苗在体内引发体液和细胞免疫反应。该项目努力将细胞生物学加工和呈递的体外研究与热敏感蛋白/抗原复合物的免疫原性的体内研究结合起来，热敏感蛋白/抗原复合物是基于天然佐剂的T细胞刺激疫苗的一种迷人的新方法。

We will study the immunogenicity of antigen/hsp complexes that are either expressed (as endogenous complexes) by antigen-presenting cells (APC), or taken up by APC as exogenous complexes, processed and presented to lymphocytes. Methods for the production, purification and characterization of recombinant hsp73- fusion antigen complexes will be refined. The cell biology of processing exogenous or endogenous hsp/antigen complexes for MHC class I-restricted epitope presentation to cytotoxic T lymphocytes (CTL) will be studied. The main focus will be on the intracellular traffic of exogenous or endogenous hsp/antigen complexes, the subcellular site at which processing takes place, the site and kinetic of the generation of presentation-competent MHC class I molecules, and the proteolytic system(s) involved. Attempts will be made to facilitate proteolytic processing of hsp-bound recombinant fusion antigen by flanking the relevant epitopes with cathepsin-sensitive cleavage sites. These in vitro studies will be complemented by in vivo studies that will elucidate priming of humoral and cellular immune responses in vivo by DNA- or protein-based vaccines containing hsp/antigen complexes. The project makes an effort to integrate the in vitro study of the cell biology of processing and presentation with the in vivo study of the immunogenicity of hsp/antigen complexes that are a fascinating, novel approach of T cell-stimulating vaccines based on a natural adjuvant.