Immunogenicity of the Type IV Secretin System

项目摘要 由于它们的表面局部化和高度保守的结构，这是由于功能限制 细菌存活和宿主细胞入侵，IV型分泌系统(TFSS)蛋白是理想的靶标 针对多种细菌病原体的疫苗开发。然而，TFSS蛋白具有 作为候选疫苗几乎没有被探索过，关于保护性的信息也很少 III型分泌系统蛋白的功效也是如此。我们建议使用无浆体边缘 模型来检验这样的假设，即用天然成分组成的连接免疫原免疫 相关的TFSS膜蛋白产生保护性免疫，并依赖于 各组分之间的分子间相互作用。关联识别的要求 独特但自然复杂的蛋白质上T和B淋巴细胞表位的比例必然会不同 表达不同MHC II类等位基因的个体，因为T细胞表位的识别是 依赖MHC II类药物。通过测试表达不同II类单倍型的牛， 代表人类MHC II类表达多样性的免疫学后果 这些分子间的相互作用是可以确定的。因此，这项提议解决了两个重要差距 据我们所知。第一个是关于蛋白质是否天然存在的基本概念 结合在膜上形成一种分泌结构，如TFSS，可提供优越的 作为一个或多个蛋白质复合体给药时的免疫水平，比作为 单个蛋白质的混合物。我们知识中的第二个主要差距涉及保护功效 IV型分泌细胞器。我们建议使用边际模型来检验这一假设。 用天然结合的TFSS膜组成的连接免疫原进行免疫 蛋白质产生保护性免疫，依赖于分子间的相互作用 单个组件。具体目标是1]确定TFSS内关联识别的目标 外膜疫苗的细胞器具有不同的MHC II类基因；2]识别哪些 边缘木霉外膜和内膜组分中的TFSS蛋白与 免疫原性TFSS蛋白，包括VirB9、VirB10和CTP；以及3]决定是否接种 免疫连锁的TFSS蛋白对诱导显著更强的保护性免疫 在边际挑战模型中与单个蛋白质亚基相比的反应。本研究 将首次评估TFSS蛋白作为候选疫苗，如果我们的假设是 正确，将表明TFSS蛋白在细菌膜内的相互作用是必要的 产生保护性免疫。相关性 对于许多细菌病原体，包括细胞内难以分离的革兰氏阴性细菌 用抗生素治疗彻底消灭，没有安全有效的疫苗可用。这个 细菌膜制剂能够刺激有效的，有时是完全的保护 抗感染为鉴定这种膜的保护成分提供了理论基础 用于疫苗开发的组份。然而，全膜疫苗的发展 抗原受到表面蛋白抗原多样性的限制，要么在宿主抗原变异内，要么 菌株间的多样性。此外，保护大量遗传异质性的种群 个体需要了解T和B细胞表位在复杂蛋白质上的联系。这 这项研究将首次评估结构保守的IV型分泌系统(TFSS) 蛋白质作为候选疫苗，如果我们的假设是正确的，将表明TFSS的相互作用 细菌膜内的蛋白质是产生保护性免疫所必需的 遗传多样性的种群。该项目的成果将是普遍适用的疫苗 针对人类许多细菌性疾病的发展。

Project Summary Because of their surface localization and highly conserved structure due to functional constraints for bacterial survival and host cell invasion, type IV secretion system (TFSS) proteins are ideal targets for vaccine development against a wide range of bacterial pathogens. However, TFSS proteins have been virtually unexplored as vaccine candidates, and there is a paucity of information on protective efficacy of type III secretion system proteins as well. We propose to use the Anaplasma marginale model to test the hypothesis that immunization with a linked immunogen composed of naturally associated TFSS membrane proteins generates protective immunity and is dependent upon intermolecular interactions among the individual components. The requirement for linked recognition of T and B lymphocyte epitopes on unique but naturally complexed proteins will necessarily differ for individuals expressing different MHC class II alleles, because the recognition of T cell epitopes is MHC class II dependent. By testing cattle that express a diverse repertoire of class II haplotypes, representative of the diversity of MHC class II expression in humans, the immunological consequence of these molecular interactions can be determined. This proposal thus addresses two important gaps in our knowledge. The first is the fundamental concept of whether proteins that are naturally associated in the membrane to form a secretory structure, such as the TFSS, can provide superior levels of immunity when administered as a complex or complexes of proteins, than when given as a mixture of individual proteins. The second major gap in our knowledge involves the protective efficacy of the type IV secretion organelle. We propose to use the A. marginale model to test the hypothesis that immunization with a linked immunogen composed of naturally associated TFSS membrane proteins generates protective immunity and is dependent upon intermolecular interactions among the individual components. Specific aims are to 1] define the targets of linked recognition within the TFSS organelle for outer membrane vaccinates with diverse MHC class II genotypes; 2] identify which TFSS proteins in outer and inner membrane fractions of A. marginale are closely associated with immunogenic TFSS proteins, including VirB9, VirB10, and CTP; and 3] determine if vaccination with immunologically linked TFSS protein pairs induces significantly stronger protective immune responses as compared to individual protein subunits in an A. marginale challenge model. This study will, for the first time, evaluate the TFSS proteins as vaccine candidates and if our hypothesis is correct, will show that interaction of TFSS proteins within the bacterial membrane is necessary for generating protective immunity. Relevance For many bacterial pathogens, including intracellular gram-negative bacteria that are difficult to completely eliminate with antibiotic treatment, safe and effective vaccines are not available. The ability of bacterial membrane preparations to stimulate effective, and sometimes complete, protection against infection provides a rationale for identifying the protective components of such membrane fractions for use in vaccine development. However, development of vaccines against total membrane antigens is constrained by surface protein antigenic diversity¿either within host antigenic variation or diversity among strains. Furthermore, protecting a large population of genetically heterogeneous individuals requires understanding the linkage of T and B cell epitopes on complexed proteins. This study will, for the first time, evaluate the structurally conserved type IV secretion system (TFSS) proteins as vaccine candidates and if our hypothesis is correct, will show that interaction of TFSS proteins within the bacterial membrane is necessary for generating protective immunity in a genetically diverse population. The results of this project will be generally applicable vaccine development for many human bacterial diseases.