Molecular Characterization of C. neoformans Antibodies

描述(由申请人提供)：新生隐球菌是免疫力受损患者的主要真菌病原体，包括晚期艾滋病毒感染者、器官移植和免疫抑制疗法的患者。此外，越来越多的证据表明，这种真菌可以在人类中建立潜伏感染，这可能会对其他慢性病的发展产生深远的后果，可能包括哮喘。新生芽孢杆菌有多种毒力因子，其中多糖壳被认为是最重要的。该胶囊由至少三种成分组成，分别为葡醛酸氧甘露聚糖(GXM)、半乳糖基甘露聚糖(GalXM)和高度甘露糖化的甘露糖蛋白。对囊膜GXM的抗体反应可诱导保护性和非保护性抗体。鉴于隐球菌感染的严重性，利用体液免疫治疗和预防疾病引起了极大的兴趣。一种单抗(MAb)已经完成了初步临床研究，并正在继续开发中。与蛋白载体结合的GXM免疫可产生保护性抗体。抗体的作用机制是多因素的，包括调理、调节炎症反应和消除酵母细胞释放GXM。值得注意的是，保护性和非保护性单抗可以根据它们在体外影响GXM释放和阻止生物膜形成的能力来区分。虽然目前对抗体在体内和体外的作用机制已有很多了解，但保护性和非保护性单抗识别的GXM表位的分子性质尚不清楚。此外，抗体在酵母细胞水平上阻止多糖释放的作用机制尚不清楚。这项申请建议继续研究抗体与新生葡萄球菌衣壳的相互作用。与以前的资助周期不同的是，当努力集中在抗体分子的分子分析上时，这项建议将研究计划的重点重新放在多糖抗原和胶囊上。除了继续研究GXM的抗体外，本申请还建议探索GalXM在胶囊结构中的作用。我们提出了三个具体的目标：1)确定结合保护性和非保护性单抗的多糖分子结构(S)；2)确定保护性单抗阻止新生弧菌释放GXM的机制；3)确定GalXM对新生弧菌囊膜结构组织的贡献，并评价针对这种多糖的抗体的有效性。

DESCRIPTION (provided by the applicant): Cryptococcus neoformans is a major fungal pathogen for individuals with impaired immunity, including those with advanced HIV infection, organ transplants, and on immunosuppressive regimens. Furthermore, there is increasing evidence that this fungus can establish latent infection in humans that could have profound consequences for the development of other chronic diseases, including possibly asthma. C. neoformans has several well-characterized virulence factors, among which a polysaccharide capsule is considered to be the most important. The capsule is composed of at least three components known as Glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and highly mannosylated proteins known as mannoproteins. Antibody responses to the capsular GXM elicit protective and non-protective antibodies. Given the seriousness of cryptococcal infections there has been great interest in harnessing humoral immunity for therapy and prevention of disease. A monoclonal antibody (mAb) has completed preliminary clinical studies and continues in development. Immunization with GXM conjugated to protein carriers elicits protective antibodies. The mechanism of antibody action is multifactorial and includes opsonization, modulation of the inflammatory response, and abrogation of GXM release from yeast cells. Remarkably, protective and non- protective mAbs can be distinguished by their ability to affect GXM release and block biofilm formation in vitro. Although much is now known about the mechanism of antibody action in vivo, and in vitro, the molecular nature of the GXM epitopes recognized by protective and non-protective mAbs is unknown. Furthermore, the mechanisms of action of antibodies at the level of the yeast cell that abrogate polysaccharide release are not understood. This application proposes to continue the study of the interaction of antibodies with the capsule of C. neoformans. In contrast to the prior funding cycles when the effort was focused on molecular analysis of the antibody molecule, this proposal refocuses the research program on the polysaccharide antigen and the capsule. In addition to continuing to study antibodies to GXM this application proposes to explore the role of GalXM in capsule structure. Three specific Aims are proposed: 1) To define the polysaccharide molecular structure(s) that bind protective and non-protective mAbs; 2) To determine the mechanism by which protective mAbs prevent GXM release from C. neoformans; 3) To determine how GalXM contributes to C. neoformans capsular structure organization and evaluate the efficacy of antibodies to this polysaccharide.