Cell Mimic Microarrays for the Multivalent Pathogen Profiling & Characterization

描述(申请人提供)：该研究项目旨在开发用于病原体的定量分析和表征、病原体抑制剂的筛选和疫苗开发的流控葡聚糖微阵列。该方法针对病原体攻击初始阶段的一个共同机制：通过受体蛋白和多糖分子之间的多价相互作用识别和附着宿主细胞。多糖的巨大多样性和多价相互作用的复杂性要求使用大规模的图谱和分析技术，特别是多糖微阵列。所提出的流控方法克服了目前糖链微阵列技术的两大局限性：缺乏流动性和难以定量控制糖链密度。多价细胞表面相互作用通常需要流体细胞膜环境的流动性，并且是表面多糖密度的强大功能。为了定量地将葡聚糖微阵列应用于分析和表征，必须确保在广泛的范围内移动和控制葡聚糖密度。第二阶段的具体目标是：目标1：以流感病毒的主要抗原血凝素和HIV-1病毒上甘露糖部分的结合受体DC-SIGN为模型系统，建立二级相互作用在结合亲和力、亲和力和特异性中的作用。这些实验将确定流体微阵列在描述和表征复杂病原体-细胞表面相互作用方面的普遍适用性；目标2：使用几种对甘露糖具有不同亲和力和选择性的大肠杆菌作为模型系统，并建立流体和密度梯度多糖微阵列可用于定量描述同一物种的菌株之间结合亲和力和多价性的变异性。量化这种变异性对于理解和监测随机突变如何导致新的病原体威胁至关重要；目标3：建立优化流体多聚糖微阵列的化学程序，包括支持的脂质双分子层斑点的空间限制、斑点区域外表面的有效阻断、微阵列干燥和复水时的可恢复性以及内容物多聚糖微阵列的长期稳定性。这些实际问题必须在开发作为可行产品的流控葡聚糖微阵列时得到解决。这一研发计划的长期目标是开发一种有效的高通量工具来对抗病原体威胁。 公共卫生相关性：该研究项目旨在开发模仿糖分子微阵列的细胞膜，用于病原体的特征分析和表征，并用于筛选针对病原体的疫苗和抑制剂。)

DESCRIPTION (provided by applicant): This research project aims to develop fluidic glycan microarrays for the quantitative profiling and characterization of pathogens, and for the screening of pathogen inhibitors and the development of vaccines. The approach targets a common mechanism at the initial stage of pathogen attack: the recognition of and attachment onto host cells via multivalent interaction between receptor proteins and glycan molecules. The tremendous variation in glycans and the complexity in multivalent interaction have necessitated the use of large-scale profiling and analysis techniques, particularly glycan microarrays. The proposed fluidic approach overcomes two major limitations of current glycan microarray technology: the lack of mobility and the difficulty in quantitatively controlling glycan density. Multivalent cell surface interactions often require mobility of the fluidic cell membrane environment and are strong functions of surface glycan density. In order to quantitatively apply the glycan microarray in profiling and characterization, one must ensure mobility and control of glycan density over a broad range. The specific aims during phase-II are: Aim 1: using haemagglutinin, a predominant antigen on influenza viruses, and the dendritic cell receptor DC-SIGN, a binding receptor for mannose moieties on HIV-1 virus, as model systems and establish the roles of secondary interactions in binding affinity, avidity, and specificity. These experiments will establish the general applicability of the fluidic microarrays in profiling and characterizing complex pathogen-cell surface interactions; Aim 2: using several strains of E. coli with varying affinity and selectivity towards mannose as model systems and establishing that the fluidic and density gradient glycan microarray can be used to quantitatively profile the variability in binding affinity and multivalency among strains of the same species. Quantifying such variability is essential to the understanding and surveillance of how random mutations can lead to new pathogen threats, as exemplified by the recent outbreaks of avian flu and swine flu; Aim 3: To establish chemical procedures for the optimization of the fluidic glycan microarray, including spatial confinement of the supported lipid bilayer spots, efficient blocking of surfaces outside the spotted areas, recoverability in drying and rehydration of the microarrays, and long term stability of content glycan microarrays. These practical issues must be addressed in developing the fluidic glycan microarray as a viable product. The long-term goal of this R&D plan is to develop an effective high-throughput tool in the combat against pathogen threats. PUBLIC HEALTH RELEVANCE: This research project aims to develop cell-membrane mimicking microarrays of sugar molecules for the profiling and characterization of pathogens, and for the screening of vaccines and inhibitors against pathogens. )