Identification of Ehrlichia ruminantium proteins that activate cellular immune responses using a reverse vaccinology strategy

Abstract

Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-ᵧ) production in E. ruminantium–immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-ᵧ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.