Immunogenicity of the Type IV Secretin System

描述（由申请人提供）：IV型分泌系统（TFSS）蛋白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入侵的功能限制），是开发针对多种细菌病原体的疫苗的理想靶标。然而，TFSS蛋白作为候选疫苗实际上尚未被探索，并且关于III型分泌系统蛋白的保护功效的信息也很缺乏。我们建议使用边际无原体模型来验证这样的假设，即由天然相关的TFSS膜蛋白组成的免疫原免疫产生保护性免疫，并依赖于单个组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，T细胞表位和B淋巴细胞表位在独特但自然复杂的蛋白上的关联识别需求必然不同，因为T细胞表位的识别依赖于MHC II类。通过测试表达多种II类单倍型的牛，可以确定这些分子相互作用的免疫学后果，这些单倍型代表了人类MHC II类表达的多样性。因此，这一建议解决了我们知识中的两个重要空白。首先是一个基本的概念，即在膜中自然结合形成分泌结构的蛋白质，如TFSS，作为一种或多种蛋白质复合物给药时，是否比作为单个蛋白质的混合物给药时能提供更高水平的免疫力。我们知识中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 . marginale模型来验证这样的假设，即由天然相关的TFSS膜蛋白组成的免疫原免疫产生保护性免疫，并依赖于单个组分之间的分子间相互作用。具体目的是：1]确定具有不同MHC II类基因型的外膜疫苗的TFSS细胞器内链接识别的靶标；2]鉴定毛豆外膜和内膜中哪些TFSS蛋白与免疫原性TFSS蛋白密切相关，包括VirB9、VirB10和CTP；[3]确定接种免疫相关的TFSS蛋白对是否比接种边缘芽孢杆菌攻击模型中的单个蛋白亚基更能诱导保护性免疫反应。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，将表明细菌膜内TFSS蛋白的相互作用对于产生保护性免疫是必要的。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，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保护的能力，为确定这种膜组分的保护成分以用于疫苗开发提供了依据。然而，针对全膜抗原的疫苗开发受到表面蛋白抗原多样性的限制——无论是宿主内的抗原变异还是菌株间的抗原多样性。此外，保护大量遗传异质性个体需要了解T和B细胞表位在复杂蛋白上的联系。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对于在遗传多样性人群中产生保护性免疫是必要的。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.