The long term goal of this research project is to understand the role of ehrlichial glycoproteins in protective immunity to Ehrlichia chaffeensis. Human monocytotropic ehrlichiosis (HME) is a life-threatening emerging tick-borne zoonosis caused by the obligately intracellular bacterium, E. chaffeensis. Antibody plays a substantial role in immunity to E. chaffeensis, but the protective antigens and epitopes involved in the development of a protective antibody response remain relatively unknown. Several glycoproteins have been identified in E. chaffeensis that elicit strong antibody response including the gp120, gp200 and gp47, and we have determined that glycans are important epitope determinants that contribute substantially to the development of glycoprotein specific antibody. The objectives of this proposal are to molecularly define the E. chaffeensis gp47 glycopeptide epitopes, and to determine the role of glycoprotein-specific antibodies in immunity. We hypothesize that many of the molecular determinants that elicit protective antibodies to E. chaffeensis are defined by O-linked hexose glycopeptides within major immunoreactive glycoproteins, including gp47. We propose the following specific aims to test this central hypothesis: 1) define the E. chaffeensis gp47 glycan compostion, linkage and attachment site(s), 2) determine the in vivo protective efficacy of gp47 glycopeptide antibodies, and 3) determine the contribution of the glycoproteome in antibody recognition and protection against E. chaffeensis infection. The gp47 glycan composition, linkage, and attachment site(s) will be determined by gas chromatography and mass spectrometry. The role of a major glycopeptide antibody epitope present in the gp47 in antibody mediated immunity to E. chaffeensis will be determined by in vivo efficacy experiments using a SCID mouse model of E. chaffeensis infection to determine immunity provided by anti-glycopeptide and anti-peptide antibodies. The role of the E. chaffeensis glycoproteome in development of protective antibodies will be determined in a SCID mouse model to evaluate protection provided by polyclonal antibodies produced against native and glycan-altered E. chaffeensis. The proposal aims to further the limited understanding of ehrlichial glycoproteins and their role in immunity to E. chaffeensis. HME is an emerging public health concern, and this research will facilitate rational development of vaccines and therapeutics against the most prevalent life-threatening tick- borne disease in North America.