Characterization of Antigen Specific Cytotoxic T-cells to Theileria parva: Evaluation of Biomarkers and Cytotoxicity Potential

Abstract

Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle leading to major economic losses in Eastern and Southern Africa including Kenya. A live vaccine against the parasite exists normally administered by subcutaneously injecting live parasites simultaneously with long acting oxytetracycline antibiotic. Nevertheless, challenges such as requirement of cold chain storage, difficult production procedures and the live vaccine being potentially lethal, makes it vital to explore other vaccine alternatives including inactivated vaccines and recombinant subunit vaccines. This study seeks to provide scientific output that will help in continued effort to develop new vaccine strategies. The mainstay of this exploration is a much better understanding of the CD8+ T-cells which are believed to be the main line of defence in East Coast fever. Hence it is essential to generate a profile of the T. parva specific CD8+ killer cells. In this study the phenotypic and functional characterization of these T. parva specific CD8+ Cells was determined by; i) Expression of surface and intracellular effector biomarkers by FACS analysis, ii) Degree of cell activation by secretion of interferon γ by ELISPOT analysis, and iii) Cytotoxic potential of CD8+ T-cells against T. parva infected cells was done in comparison to γδ T-cells. Blood samples were taken from the A18 BoLA type experimental cattle immunized with the live sporozoite vaccine. Bulk CTL and purified CTL cell lines were then generated from the experimental animals. From this study various surface and intracellular biomarkers found on T. parva specific CD8+ T-cells were identified. These included; CD2, CD3, CD4, CD25, CD44, CD45RO, CD62L, CD71, WC1, γδTCR, Perforin, FasL. Two of these markers indicated the presence of another T cell subsets γδ T-cells (WC1 and γδ TCR). It was observed from three cell lines (BF076, BF077, and BF079) that the degree of activation of CD8+ T-cells to T. parva antigen Tp1214-224 as compared to γδ T-cells was not significantly different (P > 0.05). The CD8+ T-cells exhibited significant cytotoxicity to T. parva infected lymphocytes while the γδ T-cells exhibited no measurable cytotoxicity. The study revealed that two of the activation markers, perforin and CD25 are possible surrogate markers for cytotoxicity, which is known to correlate with immunity to ECF infection. This study provided additional tools for characterizing cell-mediated immunity to T. parva induced by recombinant parasite antigens.