Several studies have shown that cytotoxic T lymphocytes (CTL) require CD4 + Th1 epitopes to generate strong immune responses to intracellular pathogens. However， not much is known about Ehrlichia ruminantium epitopes， particularly those that can be considered potential candidates for inclusion in a multi-epitope vaccine. In order to identify CD4 Th1 epitopes that induce IFNγ， a number of proteins previously identified as immunogenic were first screened to determine if they induce cellular immunity in tick infected immune sheep PBMC. Significant IFN-γ production and other Th1 cytokines were evident for 10 recombinant proteins in all sheep tested. Secondly， peptides (n = 246) derived from the to... More
Several studies have shown that cytotoxic T lymphocytes (CTL) require CD4 + Th1 epitopes to generate strong immune responses to intracellular pathogens. However， not much is known about Ehrlichia ruminantium epitopes， particularly those that can be considered potential candidates for inclusion in a multi-epitope vaccine. In order to identify CD4 Th1 epitopes that induce IFNγ， a number of proteins previously identified as immunogenic were first screened to determine if they induce cellular immunity in tick infected immune sheep PBMC. Significant IFN-γ production and other Th1 cytokines were evident for 10 recombinant proteins in all sheep tested. Secondly， peptides (n = 246) derived from the top 10 E. ruminantium vaccine candidate proteins were assayed using enzyme linked immunospot (ELISPOT) assay， quantitative real-time PCR and flow cytometry. Of the 246 peptides， 23 peptides， Erum0660 (p0660-42)， Erum1150 (p1150-18， p1150-19)， Erum2540 (p2540-6， p2540-16， p2540-19， p2540-20， p2540-21)， Erum5420 (p5420-13， p5420-14)， Erum7140 (p7140-6， p7140-7， p7140-12， p7140-13， p7140-20)， Erum7320 (p7320-8， p7320-9， p7320-21)， Erum7350 (p7350-9)， Erum7360 (p7360-8)， Erum7620 (p7620-2， p7620-12) and Erum8010 (p8010-8) were identified that stimulate the best and different cell mediated immune responses. Amino acid sequences of these peptides except for p7140-12， p7140-13， p7140-20， and p7350-9 were conserved between 13 different local strains. These peptides could efficiently induce memory CD4 T cells to rapidly proliferate and significantly increase IFN-γ production in immune sheep PBMC. The upregulation of pro-inflammatory cytokines， which include， IL-1α， IL-2， IL-12p40， TNF-α， IFN-γ， inducible nitric oxide synthase (iNOS) and granulocyte-macrophage colony stimulating factor (GM-CSF) was also detected. Our results show that these peptides could serve as promising candidates for a multi-epitope vaccine against E. ruminantium.
