Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses.

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle.

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.

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

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.

Immunisation of cattle against heartwater by infection with Cowdria ruminantium elicits T lymphocytes that recognise major antigenic proteins 1 and 2 of the agent.

There is growing evidence that immunity of cattle to Cowdria ruminantium infection is mediated by T lymphocytes. C. ruminantium antigens that stimulate these responses are therefore of considerable importance to the development of a sub-unit vaccine against the disease. We have examined T cell responses against recombinant analogues of the surface-exposed C. ruminantium major antigen 1 (MAP1) a 28.8 kDa protein and MAP2 (21 kDa) antigen in cattle immunised by infection and treatment. Vigorous and sustained proliferative responses to both antigens were observed in peripheral blood mononuclear cells from immune cattle. MAP1-specific responses were predominantly restricted to cluster of differentiation four antigen positive T cells (CD4+ T cells). Reverse transcription polymerase chain reaction (RT-PCR) analysis of cytokine expression by T cell lines derived from this population revealed strong expression of interferon gamma (IFN-gamma), interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha), tumour necrosis factor beta (TNF-beta), interleukin-2 receptor alpha (IL-2Ralpha) transcripts, and weak expression of IL-2 and IL-4. Supernatants from these T cell cultures contained IFN-gamma protein. CD4+ T cell clones specific for MAP1 were generated. Two of these clones proliferated in the presence of autologous infected endothelial cells. In contrast, the response to MAP2 was characterised largely by proliferation of gamma delta (gammadelta) T cells. RT-PCR analysis of cytokine expression by T cell lines which were dominated by gammadelta T cells revealed expression of IFN-gamma, TNF-alpha, TNF-beta, IL-2Ralpha transcripts. Supernatants of these T cell cultures also contained IFN-gamma protein. Our findings indicate that immunisation of cattle by infection with C. ruminantium results in generation of MAP1- and MAP2-specific T cell responses that may play a role in protection against the pathogen.