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.
