PHOSPHOMANNAN AS A VACCINE CANDIDATE

在当前的MRU资助期间，我们确定了由包封在脂质体中的白色念珠菌磷酸甘露聚糖复合物（PMC）材料组成的疫苗在小鼠中诱导了针对播散性念珠菌病的保护性应答。由于血清抗体和PMC中β-1，2-甘露三糖特异性的单克隆抗体而出现保护作用。我们将通过检验两个工作假设来积极地追求这些观察结果。1.脂质体疫苗可以通过利用稳定形式的脂质体和/或通过制备甘露聚糖-蛋白质缀合物消除疫苗的脂质体部分来改进。2.可以构建编码模拟关键或“保护性”甘露聚糖表位的肽（“肽模拟物”或“模拟表位”）的DNA疫苗。 这些新方法为疫苗有效性的巨大改善提供了巨大的潜力，因为可以掺入额外的组分，如念珠菌细胞壁蛋白（由Edwards和Mitchell实验室提供）。本文将通过以下具体目标对假设进行检验：1.利用稳定的脂质体构建体来改进脂质体疫苗制剂。保护性表位将共价连接到多年稳定的聚合脂质体表面。这是一种新颖的方法，但Jon Nagy博士的化学专业知识确保了脂质体可以根据需要进行快速和适当的修饰。2.构建甘露聚糖关键表位蛋白偶联物。甘露聚糖将与适当的载体蛋白偶联。这种方法将使我们能够利用广泛选择的念珠菌细胞壁蛋白的使用，这些蛋白将通过Edwards和Mitchell实验室提供。3.构建基于肽模拟表位的DNA疫苗。我们实验室正在鉴定模拟保护性甘露聚糖表位的候选肽。我们将把编码模拟表位的基因克隆到合适的真核表达载体中，并确定接种疫苗的小鼠是否携带模拟表位的抗体，并保护其免受念珠菌感染。此外，将通过Edwards和Mitchell实验室的工作提供的与念珠菌细胞壁蛋白融合的肽模拟表位将成为小鼠主要溶酶体糖蛋白（lgp-A或LAMP-1）的一部分，用于转移到MHC II类抗原加工区室。布鲁斯格兰杰博士的专业知识确保了DNA构建体的快速变异。

During the current MRU funding period, we established that a vaccine comprised of Candida albicans phosphomannan complex (PMC) material encapsulated into liposomes induced a protective response in mice against disseminated candidiasis. The protection appeared due to serum antibodies and a monoclonal antibody specific for a beta-1,2-mannotriose in the PMC was protective. We will aggressive pursue these observations by testing two working hypotheses. 1. The liposomal vaccine can be improved by utilizing a stabilized form of liposomes and/or eliminating the liposomal part of the vaccine by making mannan-protein conjugates. 2. A DNA vaccine that encodes peptides that mimic the critical or "protective" mannan epitopes ("peptide mimetics" or "mimotopes"), can be constructed. These new approaches give great potential for vast improvements in the vaccine effectiveness because additional components, such as Candida cell wall proteins (provided by the Edwards and Mitchell labs) may be incorporated. The hypotheses will be tested by the following specific aims: 1. Utilize stabilized liposomal constructs to improve the liposomal vaccine formulation. Protective epitopes will be covalently linked to the surface of polymerized liposomes that are stable for years. This is a novel approach, but the chemistry expertise of Dr. Jon Nagy ensures that the liposomes may be quickly and appropriately modified as needed. 2. Construct protein conjugates of the critical mannan epitopes. The mannans will be coupled to an appropriate carrier protein. This approach will allow us to exploit the use of a wide selection of Candida cell wall protein that will be supplied though the Edwards and Mitchell labs. 3. Construct a DNA vaccine based on peptide mimotopes. Candidate peptides that mimic protective mannan epitopes are being identified in our laboratory. We will clone genes the encode mimotopes into an appropriate eukaryotic expression vector and determine if vaccinated mice taking antibodies to the mimotopes and are protected against candidiasis. Additionally, peptide mimotopes fused to candida cell wall protein that will be supplied through the work of the Edwards and Mitchell laboratories will be made part of a mouse major lysosomal glycoprotein (lgp-A or LAMP-1) for diversion into an MHC class II antigen processing compartment. The expertise of Dr. Bruce Granger ensures the expeditious making of variations of DNA constructs.