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.