Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium

Abstract

Ehrlichia ruminantium is an obligate intracellular pathogen and, as such, cell mediated immunity plays a key role in the control of bacterial replication and subsequent protection against heartwater in ruminants. IFN-γ has been shown to be a potent inhibitor of E. ruminantium growth in endothelial cells in vitro. Thus, identification of antigens that preferentially activate T cells to proliferate and secrete IFN-γ needs to be done so that they can be evaluated as vaccine candidates. Previously, a cocktail of four open reading frames (ORFs) that induce 100% protection after needle challenge have been identified. However, only 20% protection was obtained after tick challenge in the field, when administered as a DNA vaccine in sheep. Because only limited protection was obtained during a field vaccine trial, our research focused on the identification of additional ORFs as a mean to improve the efficacy of this vaccine. Because secreted proteins are reported to be major targets in a specific immune response we hypothesize that they may be potential heartwater vaccine candidates. Five ORFs (Erum5000, Erum5010, Erum7760, Erum8060 and Erum8610) encoding secreted E. ruminantium proteins were selected from the Welgevonden stock genome sequence using bioinformatics tools. The ORFs were cloned into a pET 102/D-TOPO® vector. The corresponding recombinant (r) proteins were expressed in a bacterial expression system and the expression was confirmed by immunoblots using anti-His antibodies and sheep sera. Proteins were purified by immobilized metal ion affinity chromatography and resulted in a yield of 200 μg-2000 μg per 100 ml and 1L cultures respectively. Four of the five recombinant proteins (rErum5000, rErum7760, rErum8060 and rErum8610) could be expressed. Recombinant proteins were assayed to determine whether they induce recall cellular immune responses in vitro. The peripheral blood mononuclear cells used in the assays were obtained from a naïve and four heartwater immune sheep. Significant proliferative responses (p ≤ 0.01) were evident for 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610). IFN-γ production was determined using an ELISPOT assay and 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610) induced IFN-γ production. Each recombinant protein had its own optimum concentration for inducing immune responses and the responses differed between animals. In addition, real-time PCR was used to measure IFN-γ and IL-4 gene expression by antigen stimulated immune PBMC. The real-time PCR results correlated with the ELISPOT assay results. Based on the results obtained, it can be concluded that a Th1 type immune response was elicited. Thus these proteins that induced proliferation and IFN-γ production may be important in protection against heartwater and will be tested in future vaccine studies.