In vitro and in vivo evaluation of five low molecular weight proteins of Ehrlichia ruminantium as potential vaccine components

Low molecular weight (LMW) proteins of E. ruminantium can induce proliferation of immune peripheral blood mononuclear cells (PBMCs) and the production of interferongamma (IFN-y ) by CD4+-enriched T-cells. In this study, a reverse vaccinology approach was applied to identify additional vaccine candidates focusing on genes that encode LMW proteins smaller than 20 kDa. Five open reading frames (ORFs) were selected from the E. ruminantium genome and their corresponding recombinant (r) proteins were produced in a bacterial expression system. Their ability to induce proliferative responses and IFN-y production was evaluated in vitro using lymphocyte proliferation and ELISPOT assays. All five recombinant proteins induced proliferation of immune PBMCs and IFN-y production by these cells. The corresponding five genes were each individually incorporated into pCMViUBs, a mammalian expression vector and tested as a potential vaccine in sheep using a DNA prime - protein boost immunisation regimen. A cocktail of these DNA constructs protected one out of five sheep against a virulent E. ruminantium (Welgevonden) needle challenge. Three of the five vaccinated sheep showed an increase in their proliferative responses and production of IFN-y before challenge. This response decreased after challenge in the sheep that succumbed to the challenge and increased in the sheep that survived. This finding indicates that sustained IFN -y production is likely to be involved in conferring protective immunity against heartwater.This work was supported by the Red Meat Research and Development Trust of South Africa.http://www.sciencedirect.com/science/journal/01652427ab2013 (Author correction

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

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.The South African Department of Agriculture OV9/23/C167 grant and the FP6 EU INCO-DEV EPIGENEVAC FP6-003713 grant.http://www.elsevier.com/locate/vetimmab201