Exploiting antibody catalysis for treating Cryptococcosis

晚期HIV感染的患者有患隐球菌病的风险，这是一种破坏性的真菌感染，影响 主要是以中枢神经系统的形式难以治疗的脑膜脑炎。无数的研究 过去二十年的研究表明，抗体(Ab)介导的免疫可以做出重要贡献 来防御新生隐球菌。单抗是很有前途的治疗药物 隐球菌病的试剂。这种真菌在病原真菌中是不寻常的，因为它有一个很大的 将大量的多糖抗原释放到受感染组织中的多糖胶囊，在那里它 可干扰免疫反应，从而促进感染的持续性。组织抗原是 与免疫重建炎症综合征的发病机制有关，该综合征通常会使 同时感染HIV和隐球菌的患者与抗逆转录病毒治疗相关的免疫反弹 感染。目前，还没有从组织中去除多糖的治疗方法。我们小组发现 一些抗新生隐孢子虫胶囊的单抗具有降解胶囊多糖的催化活性。这 是一个令人兴奋的发现，因为它增加了开发一种新型隐球菌病治疗方法的可能性 基于通过去除隐球菌的被膜并降解使其易受免疫系统攻击 组织抗原沉积。这项申请提出了实验，以确定抗体介导的贡献 催化抗体介导的保护，目标是开发一种新的治疗策略 基于清除组织抗原沉积的艾滋病相关隐球菌病。隐球菌的组织清除 将在正常和免疫缺陷小鼠身上进行抗原检测，以确定炎症的贡献 以达到这种效果，并模拟艾滋病患者的组织反应。我们的方法是评估一个大型现有的 收集单抗，以确定最有效的催化抗体，并探索抗体- 介导的催化作用影响隐球菌细胞。我们将使用抗体结合位点的定点突变来 绘制催化结构域，并产生具有零催化活性的变体，这将使我们能够区分 经典的抗体函数与催化活性产生的抗体函数之间的差异。将对催化抗体进行研究 在小鼠模型中，以确定其从组织中去除多糖的能力。提出了三个目标：1) 建立抗体介导的催化作用对CN胶囊的影响；2.抗体介导的催化作用部位的确定 催化并产生具有催化活性和不具有催化活性的单抗；以及3)确定抗体- 介导的GXM水解酶..我们预计，在这项工作完成后，我们将能够部署 艾滋病相关隐球菌病患者的新型抗体疗法。

Patients with advanced HIV infection are at risk for cryptococcosis, a devastating fungal infection that affects primarily the central nervous system in the form of a difficult to treat meningoencephalitis. Numerous studies over the past two decades have shown that antibody (Ab)-mediated immunity can make a critical contribution to host defense against Cryptococcus neoformans. Monoclonal antibodies (mAbs) are promising therapeutic reagents for cryptococcosis. This fungus is unusual among pathogenic fungi in that it has a large polysaccharide capsule that releases copious amounts of polysaccharide antigen into infected tissues, where it can interfere with the immune response and thus facilitate the persistence of infection. Tissue antigens are implicated in the pathogenesis of immune reconstitution inflammatory syndrome, which often complicates the rebound in immunity associated with antiretroviral therapy in patients with concurrent HIV and cryptococcal infection. Currently, there is no therapy to remove polysaccharide from tissues. Our group discovered that some mAbs to the C. neoformans capsule have catalytic activity that degrades the capsular polysaccharide. This is an exciting finding because it raises the possibility of developing a new type of therapy for cryptococcosis based on making cryptococcal cells vulnerable to the immune system by removing their capsule and degrading tissue antigen deposits. This application proposes experiments to ascertain the contribution of Ab-mediated catalysis to Ab-mediated protection with the goal of developing a new therapeutic strategy for patients with AIDS-associated cryptococcosis based on clearance of tissue antigen deposits. Tissue clearance of cryptococcal antigen will be done in both normal and immunodeficient mice to ascertain the contribution of inflammation to this effect and mimic tissue responses in patients with AIDS. Our approach is to evaluate a large existing collection of mAbs to identify the most effective catalytic Ab and explore the mechanisms by which Ab- mediated catalysis affects cryptococcal cells. We will use site directed mutagenesis of the Ab-binding site to map the catalytic domain and to generate variants with null catalytic activity that will allow us to discriminate between classical Ab functions from those resulting from catalytic activity. Catalytic antibodies will be studied in mouse models to ascertain their capacity to remove polysaccharide from tissue. Three aims are proposed: 1) To establish the effect of Ab-mediated catalysis on the capsule of CN; 2.To identify the site of Ab-mediated catalysis and generate mAbs with and without catalytic activity; and 3) To establish the consequences of Ab- mediated GXM hydrolysis.. We anticipate that at the completion of this work we will be in a position to deploy a new type of antibody therapy for patients with AIDS-related cryptococcosis.