Sebatjane S.I., Pretorius A., Liebenberg J., Steyn H., Van Kleef M.
ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa; Department of Veterinary Tropical Diseases, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa
Sebatjane, S.I., ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa, Department of Veterinary Tropical Diseases, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa; Pretorius, A., ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa; Liebenberg, J., ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa; Steyn, H., ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa; Van Kleef, M., ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa, Department of Veterinary Tropical Diseases, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa
Low molecular weight (LMW) proteins of E. ruminantium can induce proliferation of immune peripheral blood mononuclear cells (PBMCs) and the production of interferon-gamma (IFN-γ) 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-γ production was evaluated in vitro using lymphocyte proliferation and ELISPOT assays. All five recombinant proteins induced proliferation of immune PBMCs and IFN-γ 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-γ 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-γ production is likely to be involved in conferring protective immunity against heartwater. © 2010 Elsevier B.V.
gamma interferon; plasmid DNA; recombinant protein; animal model; article; bacterial genome; bacterial infection; cell proliferation; controlled study; cytokine production; Ehrlichia ruminantium; enzyme linked immunospot assay; in vitro study; in vivo study; lymphocyte proliferation; molecular weight; nonhuman; open reading frame; peripheral blood mononuclear cell; sheep; vaccination; Animals; Bacterial Proteins; Bacterial Vaccines; Ehrlichia ruminantium; Immunization, Secondary; Interferon-gamma; Lymphocyte Activation; Molecular Weight; Open Reading Frames; Sheep; Vaccines, DNA; Bacteria (microorganisms); Ehrlichia ruminantium; Mammalia; Ovis aries