Candida in vivo expressed protein as a mannan carrier

念珠菌病在免疫功能低下的患者中是有问题的，表现为从危及生命的皮肤粘膜到威胁生命的血液播散性感染。念珠菌病的主要病原体是白色念珠菌，虽然有几种念珠菌很重要，但我们的研究将集中在白色念珠菌上。以前我们提供了保护性疫苗的概念证明，但我们拟议的工作将导致疫苗配方的根本改进。最初的疫苗使用牛血清白蛋白(BSA)作为蛋白载体连接到真菌甘露聚糖粗提物上，但计划中的改进包括用在疾病发病过程中表达的白色念珠菌表面蛋白取代BSA，以及合成与β连接的低聚甘露糖苷而不是粗甘露聚糖。令人兴奋的方面 工作是，与其他MRU项目的相互作用将使我们能够识别已知在人类念珠菌病发病过程中表达的真菌细胞壁表面蛋白，因为到目前为止，我们的所有工作都局限于这种人类疾病的小鼠模型。这项工作是由两个假设驱动的：用适当的体内(人)表达的白色念珠菌细胞壁表面蛋白取代BSA将导致对蛋白载体的保护性反应；用合成的β-连接甘露糖苷取代粗甘露聚糖部分将有利于产生保护性抗体反应。这两个假设将通过以下具体目标进行检验。1.利用IVIAT和蛋白质微阵列进行鉴定 候选真菌载体蛋白。2.通过利用细胞壁制剂的双向凝胶电泳来鉴定不需要大量N-糖基化的蛋白质，来精炼目标1中定义的候选蛋白质的数量。3.将潜在的载体蛋白共价偶联到甘露聚糖提取物上，并在小鼠身上测试其疫苗效力。4.用合成的β-甘露糖苷取代甘露聚糖提取物。这些目标的实现应导致研制一种既能预防粘膜念珠菌病，又能预防播散性念珠菌病的疫苗。

Candidiasis is problematic amongst immunocompromised patients, manifesting as disease ranging from life-compromising mucocutaneous to life-threatening hematogenously disseminated infections. The primary agent of candidiasis is Candida albicans and, although several species of Candida are important, our studies will focus on C. albicans. Previously we provided proof of concept for a protective vaccine, but our proposed work will result in essential refinement of the vaccine formulation. The original vaccine utilized bovine serum albumin (BSA) as a protein carrier coupled to a crude fungal mannan fraction, but the planned improvements include replacement of the BSA with a C. albicans surface protein expressed during pathogenesis of disease, and synthetic beta-linked oligomannosides instead of crude mannan. An exciting aspect of the work is that interaction with the other MRU projects will allow us to identify fungal cell wall surface proteins that are known to be expressed during pathogenesis of candidiasis in humans, as until now all of our work has been restricted to mouse models of this human disease. The work is driven by two hypotheses: replacement of the BSA with an appropriate in vivo (human) expressed C. albicans cell wall surface protein will lead to protective responses against the protein carrier; and, replacement of the crude mannan portion with synthetic beta-linked mannosides will favor production of protective antibody responses. The two hypotheses will be tested by the following specific aims. 1. Utilize IVIAT and protein microarrays to identify candidate fungal carrier proteins. 2. Refine the number of candidate proteins defined in aim 1 by utilizing 2-D gel electrophoresis of cell wall preparations to identify proteins without extensive N-glycosylation. 3. Covalently couple potential carrier proteins to mannan extracts and test the conjugates in mice for vaccine efficacy. 4. Replace the mannan extract with synthetic beta-mannosides. Fulfillment of these aims should lead to development of a vaccine that prevents both mucosal and disseminated forms of candidiasis.