Model Vaccines Exploiting Fungal Mannosylation

描述(由申请人提供)：来自新生隐球菌的甘露糖蛋白(MP)能激发强烈的T细胞介导的免疫反应。隐球菌MP含有丰富的丝氨酸/苏氨酸(S/T)区和N-X-S/T序列，前者是广泛的O-连接糖基化位点，后者则是N-糖基化位点。酵母上的N-连接和O-连接的多糖是末端甘露糖化的，而完全加工的哺乳动物糖蛋白很少有末端甘露糖基。树突状细胞(DC)上的甘露糖受体(MR)与MP结合，导致DC有效地摄取、加工和呈递MP的多肽片段。鉴于这些发现，我们假设可以利用甘露糖化提高免疫原性的能力来制造刺激T细胞反应的重组疫苗。为了验证这一假设，将在体外和体内测试模型抗原卵清蛋白(OVA)的不同糖基化制剂诱导MHC I类(CD8)和II类(CD4)限制性T细胞反应的能力。有3个具体目标：目标1是在细菌、酵母和哺乳动物细胞载体中产生重组OVA制剂。这样的预备物应该分别是未糖化的、末端甘露糖化的和糖基化但没有末端甘露糖基的。添加S/T富集区和N-到-Q替换将使研究O-和N-键对免疫原性的相对贡献成为可能。目标1中产生的OVA制剂将在目标2中用于检测OVA糖基化模式对体外抗原摄取、加工和呈递的影响，以及在目标3中测试各种糖基化重组蛋白在体内诱导免疫反应的效果。因此，对含有相同蛋白质核心但在糖基化性质和数量上不同的抗原的I类和11类限制性反应将进行比较。MR在DC上作为内吞受体的作用，允许有效地捕获甘露糖基化的OVA制剂，将使用MR阻滞剂和基因敲除小鼠进行研究。这些研究的完成将大大增加我们对外来糖基化模式的先天识别如何触发适应性免疫反应的知识，并可能为提高疫苗的免疫原性提供新的策略。

DESCRIPTION (provided by applicant): Mannoproteins (MP) from Cryptococcus neoformans elicit strong T cell-mediated immune responses. Cryptococcal MP contain serine/threonine (S/T)-rich regions which serve as sites for extensive O-linked glycosylation as well as N-X-S/T sequons that serve as N-glycosylation sites. N-linked and O-linked glycans on yeasts are terminally mannosylated whereas fully processed mammalian glycoproteins rarely have terminal mannose groups. Mannose receptors (MR) on dendritic cells (DC) bind to MP, resulting in the efficient uptake, processing and presentation of peptide fragments of MP by DC. Given these findings, we hypothesize that the capacity of mannosylation to increase immunogenicity can be exploited to make recombinant vaccines that stimulate T-cell responses. To test this hypothesis, the capacity of differentially glycosylated preparations of the model antigen, ovalbumin (OVA), to elicit MHC Class I (CD8) and class II (CD4)-restricted T cell responses will be tested in vitro and in vivo. There are 3 specific aims: Aim 1 is to generate recombinant OVA preparations in bacterial, yeast, and mammalian cell vectors. Such preps should be unglycosylated, terminally mannosylated, and glycosylated but without terminal mannose groups, respectively. Addition of S/T-rich regions and N-to-Q substitutions will permit study of the relative contribution of O- and N-linkages to immunogenicity. The OVA preparations generated in aim 1 will be utilized in aim 2 to examine the effects of OVA glycosylation patterns on antigen uptake, processing and presentation in vitro, and in aim 3 to test the efficacy of the various glycosylated recombinant proteins to induce an immune response in vivo. Thus, Class I and Class ll-restricted responses to antigens that contain identical protein cores but that qualitatively and quantitatively differ with regards to glycosylation will be compared. The role of MR on DC as endocytic receptors allowing for the efficient capture of mannosylated OVA preparations will be studied using blockers of the MR and with knockout mice. Completion of these studies should add significantly to our knowledge of how innate recognition of foreign glycosylation patterns triggers adaptive immune responses and could suggests novel strategies for increasing the immunogenicity of vaccines.