Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes

Abstract

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.

Fig. 1.

Targeted and random approaches to T. parva antigen identification. (A) Screening of targeted genes with T. parva-specific CTL from four cattle showed specific reaction to iSF transfected with targeted gene 5 (Tp2) as assessed by IFN-γ ELISpot. (B) Representative results of screening pools of random schizont cDNA with T. parva-specific CTL. By using the IFN-γ ELISpot assay, CTL from animal BV115 were used to screen COS-7 cells cotransfected with BoLA-N*01301 cDNA and pools of parasite cDNA to reveal positive pools. (C) Screening of resolved cDNA pools allowed the identification of five cDNA clones. (D) Confirmatory assays revealed specific reaction against three identical cDNA clones encoding Tp1. Responses are presented as mean numbers of SFC per well.

Fig. 2.

CTL activity against transfected targets. Lysis was determined against autologous iSF transfected with Tp1, Tp2, Tp4, Tp5, and Tp7 or COS-7 cotransfected with BoLA-N*00101 and Tp8. Filled circles, autologous schizont-infected cells; open triangles, Tp gene-transfected autologous iSF or COS-7; open circles, irrelevant PIM gene-transfected autologous iSF; open squares, Tp gene-transfected allogeneic iSF; filled triangles, Tp7 peptide-pulsed autologous iSF.

Fig. 3.

Antigen specificity of CD8⁺ T cell responses after challenge of T. parva-immune cattle. IFN-γ responses of ex vivo CD8⁺ T cells were measured in the presence of pools of overlapping peptides. Results are presented as medium corrected mean numbers of SFC per 1 × 10⁶ CD8⁺ T cells. Some time points were omitted because of a background >100 SFC per 1 × 10⁶ CD8⁺ T cells.

Fig. 4.

Antigen specificity of CD8⁺ T cells in vaccinated cattle. (A and C) Representative IFN-γ responses of five responder cattle illustrate reactivity to each of the antigens and are compared with a nonresponder calf. Responses presented are the medium corrected number of SFC per 1 × 10⁶ CD8⁺ T cells. (B and D) Four of the five IFN-γ responder cattle exhibited lytic responses and are compared with two nonresponder cattle. Filled squares, Tp1; open squares, Tp2; filled triangles, Tp4; open triangles, Tp5; crosses, Tp8; filled circles, schizont-infected cells; open circles, unpulsed T cell blasts.