Cellular immune responses induced <i>in vitro</i> by <i>Ehrlichia ruminantium</i> 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.

Ehrlichia ruminantium antigens and peptides induce cytotoxic T cell responses in vitro

“…To mimic natural infection, ticks experimentally infected with the Welgevonden strain [11] were fed onto three sheep (s6821, s6822, s6823). Briefly, uninfected Amblyomma hebraeum nymph ticks were infected by feeding on a sheep (s6819) that had been infected intravenously with E. ruminantium Welgevonden blood stabilate.…”

“…Overall, these results demonstrated that these peptides induced CD8 + T cells to produce IFN-γ and CTL responses were activated. All previous studies have shown an increase in CD8 + T cells but have not determined the cytotoxic activities induced by CD8 + T cells after stimulation with the recombinant proteins [11]. A total of 14 E. ruminantium genes were cloned in pCMViUBs as part of a previous study [7] and were selected for this study.…”

“…Much effort has been spent on the identification of proteins that induce strong cell-mediated immune responses characterised by CD4 + or CD8 + T cell responses. To date in our laboratory several E. ruminantium proteins have been characterised [7,8,9,10,11]. These studies have shown that T cells induce IFN-γ production in immune sheep or bovine peripheral blood mononuclear cells (PBMC) but assays done have not included the cytotoxic T cell assay to show that CD8 + T cells induce lysis of target cells.…”

“…In previous E. ruminantium vaccine development studies in our laboratory, 14 genes were selected as vaccine candidates based mainly on their ability to induce IFN-γ [8,9,10,11].…”

Transcriptomic analysis of Ehrlichia ruminantium during the developmental stages in bovine and tick cell culture

“…The use of bioinformatics tools to search for additional vaccine candidates against heartwater has been previously demonstrated . Although, some of the predicted antigens have been tested previously as cell-mediated DNA vaccines (Pretorius et al, 2007Sebatjane et al, 2010;Thema et al, 2016), these have displayed limited or no protection against a natural tick challenge. Our current approach focused on studying gene expression using transcriptome sequencing at both the developmental stage in vitro in mammalian and tick cells.…”