Strategies for generating high affinity antibodies against Gram negative bacteria

项目摘要 糖覆盖在细菌和其他病原体的外表面，通常非常 有效地保护这些有机体免受免疫系统的攻击。缺乏T细胞帮助，抗多糖 回应往往是低亲和力的。然而，低聚糖的结构复杂性使它们， 原则上，物种特异性标记的优秀候选者应该特异性免疫识别 发生。 拟议中的研究将使用一种方法来产生这种免疫反应。 对抗显性致病菌外表面发现的独特的三糖和四糖基序 革兰氏阴性杆菌铜绿假单胞菌和洋葱伯克霍尔德氏菌。这个 该方法涉及化学合成具有以下形式的感兴趣的糖链基序 生物偶联，这些分子与免疫原性病毒样颗粒通过 精确控制所用连杆的类型和位置，并使用高度活跃的 作为佐剂的自然杀伤T(NKT)细胞的激活物，这些细胞是诱导 在自然感染和疫苗中对糖类的强大免疫反应。 这种方法以前已经被证明能够产生超高亲和力和保护性 小鼠对革兰氏阳性肺炎链球菌的抗体应答。革兰氏阴性 生物体仍然是一个严峻得多的挑战；对病原体的强大免疫反应- 特定的细胞表面糖链基序可以代表一种有效的反应。这项研究建议 这里涉及创建和测试8种候选疫苗配方，而不是4种 碳水化合物图案，每个物种两个。对初级免疫反应的筛查将允许 优化最终免疫原的设计和选择，将进行详细的 学习。将进一步探索由这些试剂产生的高亲和力抗体，以确定 它们的结构、结合亲和力和动力学、胚系突变和保护能力 在相关的小鼠模型中对抗病原体攻击。 这一努力的成功将为人类的发展确定巨大的潜力 针对这些微生物的疫苗，并将提供信心，即总体方法可以 对其他以葡聚糖为基础的疫苗有用。

Project Summary Sugars cover the outer surfaces of bacteria and other pathogens and are usually very effective at shielding these organisms from the immune system. Lacking T cell help, anti-glycan responses are often of low affinity. Yet the structural complexity of oligosaccharides make them, in principle, excellent candidates for species-specific markers should specific immune recognition occur. The proposed research will use an approach for the generation of such immune responses against unique tri- and tetrasaccharide motifs found on the outer surfaces of dominant pathogenic strains of the Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cepacia. The method involves the chemical synthesis of the glycan motifs of interest in forms amenable to bioconjugation, the attachment of those molecules to immunogenic virus-like particles with precise control of the type and placement of the linkages used, and the use of a highly active activator of natural killer T (NKT) cells as adjuvant, these cells being central to the elicitation of robust immune responses against sugars in both natural infections and vaccines. This approach has been previously shown to generate ultra-high-affinity and protective antibody responses in mouse against Gram-positive Streptococcus pneumoniae. Gram-negative organisms remain a significantly tougher challenge; a potent immune response against pathogen- specific cell-surface glycan motifs could represent an effective response. The studies proposed here involve the creation and testing of eight candidate vaccine formulations against four carbohydrate motifs, two for each species. Screening of primary immune response will allow for optimization of the design and selection of final immunogens that will be carried into detailed studies. High-affinity antibodies generated from these agents will be further explored to determine their structures, binding affinities and kinetics, mutations from germ line, and abilities to protect against pathogen challenge in a relevant mouse model. Success in this endeavor would identify great potential for the development of human vaccines against these organisms and would provide confidence that the overall approach could be useful for other glycan-based vaccines.