Identification of CD8 T-lymphocyte epitopes in OmpB of Rickettsia conorii

The 1.2-kb DNA fragment of the Rickettsia conorii outer membrane protein B gene (OmpB_451-846) was subcloned using site-specific PCR primers and expressed as six smaller fragments: OmpB_458-652, OmpB_595-744, OmpB_595-654, OmpB_654-692, OmpB_689-744, and OmpB_739-848. NCTC cells transfected with a mammalian expression vector expressing the fragments OmpB_689-744 and OmpB_739-848 stimulated immune anti-R. conorii CD8 T lymphocytes, suggesting the presence of CD8 T-lymphocyte-stimulating epitopes on these fragments. In order to further characterize the CD8 T-lymphocyte-stimulatory elements, CD8 T-lymphocyte epitopes on OmpB_689-744 and OmpB_739-848 were mapped by overlapping synthetic peptides. The ability of these synthetic peptides to stimulate immune CD8 T lymphocytes was determined by gamma interferon (IFN-γ) production and cell proliferation after incubation with simian virus 40-transformed murine vascular endothelial cells in the presence of a 20 μM solution of each synthetic peptide. Five synthetic peptides, SKGVNVDTV (OmpB_708-716), ANVGSFVFN (OmpB_735-743), IVSGTVGGQ (OmpB_749-757), ANSTLQIGG (OmpB_789-797), and IVEFVNTGP (OmpB_812-820), induced secretion of IFN-γ at significantly higher levels than the controls. Three of these five peptides, SKGVNVDTV (OmpB_708-716), ANSTLQIGG (OmpB_789-797), and IVEFVNTGP (OmpB_812-820), also stimulated the proliferation of immune CD8 T lymphocytes. Significantly higher levels of specific cytotoxic T-lymphocyte killing were observed with the same three synthetic peptides, SKGVNVDTV (OmpB_708-716), ANSTLQIGG (OmpB_789-797), and IVEFVNTGP (OmpB_812-820).