Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/3/2004
Publication Date: 3/5/2005
Citation: Staska, L.M., Davies, C.J., Brown, W.C., Mcguire, T.C., Suarez, C.E., Park, J.Y., Mathison, B.A., Abbott, J.R., Baszler, T.V. 2005. Identification of vaccine candidate peptides in the ncsrs2 surface protein of neospora caninum by using cd4+ cytotoxic t lymphocytes and gamma interferon-secreting t lymphocytes of infected holstein cattle. Infection and Immunity. 73(3):1321-1329. Interpretive Summary: In this work we describe the identification of vaccine-candidate peptides mapped from the NcSRS2 surface protein of Neospora caninum. The peptides induced CD4+ cytotoxic and IFN-' -secreting T-lymphocites from N. caninum infected Holstein cattle. Importantly, we identified an immunodominant region in the NcSRS2 surface protein that was recognized by cattle bearing different MHC haplotypes. These findings support investigation of subunit N. caninum'vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-'-secreting T lymphocytes in cattle with varied MHC genotypes.
Technical Abstract: Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD-4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T lymphocyte responses for vaccine development against bovine neosporosis, we targeted tachizoite major surface proteins NcSAG1 and NcSRS2. In whole tachyzoite antigen-expanded bovine T lymphocyte lines recombinant NcSRS2 induced potent memory CD4+ and CD8+ T lymphocyte activation, as indicated by proliferation and IFN-' secretion, while recombinant NcSAG1 induced minimal memory response. Subsequently, T lymphocyte epitope-bearing peptides of NcSRS2 were mapped using overlapping peptides covering the entire NcSRS2 sequence. Five experimentally infected cattle with six MHC class II haplotypes were the source of immune cells to identify NcSRS2 peptides presented by common Holstein MHC haplotypes. NcSRS2 peptides were mapped using IFN-' secretion by rNcSRS2-stimulated, short term T lymphocyte cell lines, IFN-' ELISPOT assay using PBMC, and 51Cr-release cytotoxicity assay of NcSRS2-stimulated effector cells. Four of five N. caninum infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-' ELISPOT and in vivo by measuring T lymphocyte IFN-' production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 135-155 was recognized by CD4+ T lymphocytes from four of five cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-'-secreting T lymphocytes in cattle with varied MHC genotypes.