Extensive Polymorphism and Evidence of Immune Selection in a Highly Dominant Antigen Recognized by Bovine CD8 T Cells Specific for Theileria annulata

Abstract

Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines. CD8 T cells have been shown to play a key role in immunity to a variety of intracellular pathogens (65), including a number of protozoan parasites (33,36,38,55). A characteristic feature of CD8 T cell responses is that, in individual hosts, they are often directed against a few dominant epitopes (67). Consequently, mutations in sites encoding these epitopes can result in escape from immune recognition. This is well established as an important phenomenon in infections with some RNA viruses that exhibit a high rate of mutation, most notably, HIV-1 (18, 26, 39). Parasite strain-restricted CD8 T cell responses have also been reported for several protozoan infections, including human malaria and theileriosis in cattle (15,17,35). In the case of Theileria parva, variation between animals in the strain specificity of CD8 T cell responses has been shown to correlate with incomplete cross-protection between parasite strains (54). These observations suggest that polymorphism of the target parasite antigens may have arisen as a result of CD8 T cell imposed immune selection.The bovine tick-borne parasites Theileria parva and T. annulata infect and transform leukocytes, causing acute lymphoproliferative diseases that result in high levels of mortality and heavy production losses (25)....

Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery

“…Further, the BoLA-T2C restricted epitope FAQSLVCVL and the BoLA-3*00101 (AW10) restricted epitope TGASIQTTL were both found to cross-bind to BoLA-3*00201 (JSP.1) and the BoLA-T5 restricted epitope SKADVIAKY was found to bind with an affinity of 15 nM and a stability of 18 h to BoLA-1*02301 (D18.4). The latter observation confirms what has been reported earlier (MacHugh et al 2011; Svitek et al 2014). …”

Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle

“…Indeed, prior studies on the malaria parasite, P. falciparum suggest that the spleen plays an important role in the sequestration of erythrocytes containing the parasite (Contamin et al, 2000), with different parasite variants inducing the expression of alternate antigens on the erythrocyte cell surface, thereby allowing the parasite to avoid splenic destruction (Borst et al, 1995). While the role of MHC in immune modulation of T. orientalis has not been confirmed, in T. parva and T. annulata infection, CD8 + T cell responses are directed against class I MHC-bound antigenic peptides and display a clear dominance hierarchy (MacHugh et al, 2009(MacHugh et al, , 2011. T cell responses are restricted further by the specific MHC haplotypes of host cattle and in T. parva infection, the majority of these responses are focussed on a single antigenic epitope (MacDonald et al, 2010).…”

Identification of immediate early gene products of bovine herpes virus 1 (BHV-1) as dominant antigens recognized by CD8 T cells in immune cattle

“…The IFNg concentration was then calculated by correlation to the standard curve generated by the IFNg standards. In the remaining epitopemapping experiments, IFNg release by T cells was measured using a biological assay performed as described previously [53], based on the ability of the IFNg in culture supernatants to up-regulate surface expression of MHC class II on Madin-Darby bovine kidney (MDBK) cells. In preliminary studies, we showed that this assay had slightly greater sensitivity than the ELISA (data not shown).…”

“…Antigen screening was carried out essentially as described previously [53], and was based on measurement of interferon gamma (IFNg) release by CD8 T-cell lines incubated with COS-7 cells transfected with individual BHV-1 genes along with a pool of cDNAs encoding the class I heavy chains expressed by the respective animals. COS-7 cells were plated at 2Â10 4 cells per well in 96-well flat-bottom plates in Dulbecco's modified Eagle's medium (DMEM) with 10 % foetal bovine serum, 2 mM L-glutamine, 100 u penicillin and 10 µg streptomycin/ml.…”

“…First, cloned fragments of the positive cDNAs obtained by PCR amplification were screened for recognition, as described for the full-length cDNAs, in order to localize the epitopes to one discrete region. Overlapping synthetic peptides (17mers overlapping by 12 residues; Pepscan Ltd, Netherlands) representing the positive region were then screened to identify the epitope using T. annulata-infected cells as antigen-presenting cells (APC), as described previously [53]. Assays were conducted in 96-well flat-bottomed plates using 2Â10 4 CD8 T cells/well, with 10 4 peptide-pulsed APC, in a total volume of 200 ul and incubated for 48 h at 37 C. Supernatants were assayed for IFNg as a measure of recognition by the CD8 T-cell line.…”