Cellular immune responses induced in vitro by Ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides

Abstract

Secreted proteins are reported to induce cell-mediated immunity characterised by the production of interferon-gamma (IFN)-γ. In this study three open reading frames (ORFs) (Erum8060, Erum7760, Erum5000) encoding secreted proteins were selected from the Ehrlichia ruminantium (Welgevonden) genome sequence using bioinformatics tools to determine whether they induce a cellular immune response in vitro with mononuclear cells from needle and tick infected animals. The whole recombinant protein of the three ORFs as well as four adjacent fragments of the Erum5000 protein (Erum5000A, Erum5000B, Erum5000C, Erum5000D) were successfully expressed in a bacterial expression system which was confirmed by immunoblots using anti-His antibodies and sheep sera. These recombinant proteins were assayed with immune sheep and cattle peripheral blood mononuclear cells (PBMCs), spleen and lymph node (LN) cells to determine whether they induce recall cellular immune responses in vitro. Significant proliferative responses and IFN-γ production were evident for all recombinant proteins, especially Erum5000A, in both ruminant species tested. Thus overlapping peptides spanning Erum5000A were synthesised and peptides that induce proliferation of memory CD4+ and CD8+ T cells and production of IFN-γ were identified. These results illustrate that a Th1 type immune response was elicited and these recombinant proteins and peptides may therefore be promising candidates for development of a heartwater vaccine.

FIGURE 1

IFN-γ production by peripheral blood mononuclear cells stimulated with 16 mer overlapping peptides of Erum5000A.

FIGURE 2

Percentage of cells expressing CD4+CD45RO+ (a) and CD8+CD45RO+ (b) surface markers after peripheral blood mononuclear cells stimulation with Erum5000A peptides for 48 h in vitro.

FIGURE 1-A1

Western blot analysis of the whole protein rErum5000i and four fragments, Erum5000A, Erum5000B, Erum5000C and Erum5000D, Erum7760 and Erum8060. The purified recombinant proteins are expressed and purified using Champion^TM pET Directional TOPO^® expression system. Recombinant proteins were detected by using (a) immune sheep sera and (b and c) anti-His antibodies.