Immunogenicity of the Type IV Secretin System

描述（由申请人提供）：由于其表面定位和高度保守的结构（由于细菌存活和宿主细胞侵入的功能限制），IV型分泌系统（TFSS）蛋白是针对广泛细菌病原体的疫苗开发的理想靶标。然而，TFSS蛋白作为疫苗候选物几乎未被探索，并且关于III型分泌系统蛋白的保护功效的信息也很少。我们建议使用边缘无形体模型来检验这一假设，即免疫与自然相关的TFSS膜蛋白组成的免疫原产生保护性免疫，并依赖于各个组成部分之间的分子间相互作用。对于表达不同MHC II类等位基因的个体，对独特但天然复合的蛋白质上的T和B淋巴细胞表位的连锁识别的要求必然不同，因为T细胞表位的识别是MHC II类依赖性的。通过测试表达不同的II类单倍型库的牛，代表人类中MHC II类表达的多样性，可以确定这些分子相互作用的免疫学后果。因此，这一建议填补了我们知识中的两个重要空白。第一个是基本概念，即在膜中天然结合以形成分泌结构的蛋白质，如TFSS，当作为一种或多种蛋白质复合物给药时，是否可以提供比作为单个蛋白质的混合物给药时上级水平的免疫力。我们知识的第二个主要空白涉及IV型分泌细胞器的保护功效。我们建议使用A。边缘模型来检验以下假设：用由天然相关的TFSS膜蛋白组成的连接的免疫原免疫产生保护性免疫，并且依赖于各个组分之间的分子间相互作用。具体目标是1]确定不同MHC II类基因型外膜接种动物TFSS细胞器内的连锁识别靶标; 2]鉴定A.边缘亚基与免疫原性TFSS蛋白，包括VirB 9、VirB 10和CTP密切相关;和3]确定与A中的单个蛋白亚基相比，用免疫学连接的TFSS蛋白对接种是否诱导显著更强的保护性免疫应答。边缘挑战模型这项研究将首次评估TFSS蛋白作为候选疫苗，如果我们的假设是正确的，将表明TFSS蛋白在细菌膜内的相互作用是产生保护性免疫所必需的。公共卫生相关性：对于许多细菌病原体，包括难以用抗生素治疗完全消除的细胞内革兰氏阴性菌，目前还没有安全有效的疫苗。细菌膜制剂刺激有效的，有时是完全的，抗感染的能力，为鉴定用于疫苗开发的这种膜组分的保护性组分提供了理论基础。然而，针对总膜抗原的疫苗的开发受到表面蛋白抗原多样性的限制-无论是宿主抗原变异还是菌株之间的多样性。此外，保护大量遗传异质性个体需要理解复合蛋白上T和B细胞表位的连接。这项研究将首次评估结构保守的IV型分泌系统（TFSS）蛋白作为候选疫苗，如果我们的假设是正确的，将表明TFSS蛋白在细菌膜内的相互作用是在遗传多样性人群中产生保护性免疫所必需的。该项目的结果将普遍适用于许多人类细菌性疾病的疫苗开发。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH 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 to vaccine development for many human bacterial diseases.