This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In this project we wish is to establish a rhesus macaque model for the purpose of investigating the diversity of T-cell types that are elicited in response to rickettsial infection. This is an important step in our long-term goal of developing an effective vaccine against Rickettsiae. The rhesus macaque (Macaca mulatta) model is widely used in vaccine development and in studies of pathogenesis of a variety of infectious agents. Our approach will be to inoculate the animals with infectious Rickettsia parkeri organisms and examine the cell-mediated immune response. Specific objectives include: 1) Characterize cell populations that are activated in the peripheral blood mononuclear cell population (PBMC) and in the draining lymph nodes (LN) of rhesus monkeys inoculated with rickettsiae. Preferentially, we will look at the CD4(+) and CD8(+) T cell response, and catalog their cytokine profile using capture ELISA. 2) Identify CD4(+) and CD8(+) T-cell immunodominant target epitopes. We have generated over 200 rickettsia specific peptides from a consensus sequence of a major Rickettsial surface protein (OmpA). These peptides will be tested for CD4(+) and CTL immunodominant epitopes. T cell epitopes restricted to Mamu-A*01 (MHC-I) and Mamu-DR*W201 (MHC-II) will be selected using ELISPOT and intracytoplasmic cytokine staining assays. This information will contribute to the construction of Mamu-DR*W201/peptide (MHC-II) and Mamu-A*01/peptide (MHC-I) tetramers. Four animals were assigned to the project (# 3413). Blood samples were collected from these subjects and used to optimize the concentrations of the fluorochrome-conjugated antibodies and to validate the flow cytometry staining panels designed to cover large aspect of cellular immunity of Rhesus macaques during rickettsial infection.
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