MHC class II-restricted responses mediated by CD4+ T lymphocytes are central to immune control of the tick-borne pathogens of the Family Anaplasmataceae. Priming and expansion of CD4+ T lymphocytes is required for development of high affinity neutralizing IgG antibodies directed against the bacterial surface and for efficient phagocyte activation leading to bacterial killing. The goal of the research is to identify the pathogen proteins that induce the T cell responses required for immunity. To date, only four T cell immunostimulatory molecules have been identified in Anaplasma marginale, the type species, and none in the related human pathogens within the Anaplasmataceae. We will address this knowledge gap using a comprehensive strategy to identify outer membrane proteins that prime naive CD4+ T lymphocytes and induce recall memory T cell responses of immunized and protected animals. In part 1 of the project, we propose to use a combination of biochemical fractionation of outer membranes combined with genomic analysis to identify the targets of the T cell response. At the conclusion of part 1, we expect to have identified novel outer membrane proteins bearing CD4+ T cell epitopes conserved among A. marginale strains. The presence of molecular and immunological orthologs among species in the Family Anaplasmataceae indicates that the research results should be broadly applicable and enhance understanding and control of human diseases. ? ? In part 2 of the project, we will examine the requirement for linked recognition of covalently associated outer membrane proteins in generating immunity. A. marginale outer membrane proteins are covalently linked by extensive disulfide bonding. How this covalent bonding affects generation of immunity is unknown. The observation that Major Surface Protein (MSP) 1a-specific CD4+ T cells provide help for B-lymphocytes to secrete antibody to the covalently bound MSPlb suggests that the native association of outer membrane proteins can be critical in inducing protective responses. We hypothesize that this """"""""linked recognition"""""""" of CD4+ T cell and B cell epitopes from different outer membrane proteins can be mimicked using chimeric multiple epitope constructs. At the conclusion of part 2, we expect to have determined if linked recognition between covalently bound outer membrane proteins, using MSPla and MSPlb as a model complex immunogen, can be represented by a chimeric epitope construct. ? ?
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