Model Vaccines Exploiting Fungal Mannosylation

利用真菌甘露糖基化的模型疫苗

• 批准号: 7579547
• 负责人: Stuart Michael Levitz
• 金额: $ 5.52万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-20 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7579547
• 关键词: Amino Acids; Antigens; Asparagine; Binding; CD4 Positive T Lymphocytes; CD8B1 gene; Cathepsins; Cell Line; Cell-Free System; Cells; Chinese Hamster; Chinese Hamster Ovary Cell; Class; Core Protein; Cryptococcus neoformans; Cytotoxic T-Lymphocytes; Dendritic Cells; Escherichia coli; Glutamine; Glycoproteins; Human; Immune response; In Vitro; Knockout Mice; Knowledge; Label; Link; MHC Class I Genes; MHC Class II Genes; Mammalian Cell; Mannose; Measures; Modeling; Mus; N-Glycosylation Site; Ovalbumin; Ovary; Pattern; Peptide Fragments; Physiologic pulse; Polysaccharides; Preparation; Process; Proteins; Proteolysis; Pulse taking; Recombinant Proteins; Recombinant Vaccines; Recombinants; Relative (related person); Role; Serine; Site; T-Lymphocyte; Testing; Threonine; Transgenic Organisms; Vaccinated; Vaccines; Yeasts; antigen processing; base; cell mediated immune response; cell type; cytokine; glycosylation; immunogenicity; in vivo; intradermal injection; mannoproteins; mannose receptor; multicatalytic endopeptidase complex; novel strategies; prototype; receptor; response; trafficking; tumor; uptake; vector; yeast protein

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-liked 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 (CDS) 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.

新型隐球菌甘露糖蛋白（MP）可诱导强烈的T细胞介导的免疫应答。 隐球菌MP含有富含丝氨酸/苏氨酸（S/T）的区域，这些区域作为广泛的O-样 糖基化以及作为N-糖基化位点的N-X-S/T序列子。N-连接和O-连接聚糖 酵母上的糖蛋白是末端甘露糖基化的，而完全加工的哺乳动物糖蛋白很少具有 末端甘露糖基团。树突状细胞（DC）上的甘露糖受体（MR）与MP结合，导致 DC对MP的肽片段的有效摄取、加工和呈递。鉴于这些发现，我们 假设可以利用甘露糖基化增加免疫原性的能力， 刺激T细胞反应的重组疫苗。为了验证这一假设， 模型抗原卵清蛋白（OVA）的糖基化制剂，以引发MHC I类（CDS）和II类（CDS） 将在体外和体内测试（CD 4）限制性T细胞应答。 有3个具体目标：目标1是在细菌中产生重组OVA制剂， 酵母和哺乳动物细胞载体。这样的制备物应该是未糖基化的，末端甘露糖基化的， 糖基化但不含末端甘露糖基团。添加S/T富集区和N-至-Q 取代将允许研究O-和N-键对免疫原性的相对贡献。的OVA 目的1中产生的制剂将用于目的2，以检查OVA糖基化的影响 体外抗原摄取、加工和呈递的模式，并在目标3中测试 各种糖基化重组蛋白以诱导体内免疫应答。因此，I类 和对含有相同蛋白质核心的抗原的II类限制性应答， 将比较关于糖基化的定量差异。MR对DC作为内吞因子的作用 将使用阻断剂研究允许有效捕获甘露糖基化OVA制剂的受体 和基因敲除小鼠。完成这些研究将大大增加我们对以下方面的了解： 外源糖基化模式的先天识别如何触发适应性免疫应答， 提出了增加疫苗免疫原性的新策略。