Original Research

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium

Tlou A. Ngoepe, Alri Pretorius, Helena C. Steyn, Mirinda van Kleef
Onderstepoort Journal of Veterinary Research | Vol 90, No 1 | a2070 | DOI: https://doi.org/10.4102/ojvr.v90i1.2070 | © 2023 Tlou A. Ngoepe, Alri Pretorius, Helena C. Steyn, Mirinda van Kleef | This work is licensed under CC Attribution 4.0
Submitted: 02 August 2022 | Published: 23 March 2023

About the author(s)

Tlou A. Ngoepe, Department of Immunology, Agricultural Research Council-Onderstepoort Veterinary Research, Pretoria, South Africa; and, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa; and, Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria,, South Africa
Alri Pretorius, Department of Immunology, Agricultural Research Council-Onderstepoort Veterinary Research, Pretoria, South Africa; and, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa
Helena C. Steyn, Department of Immunology, Agricultural Research Council-Onderstepoort Veterinary Research, Pretoria, South Africa
Mirinda van Kleef, Department of Immunology, Agricultural Research Council-Onderstepoort Veterinary Research, Pretoria, South Africa; and, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa

Abstract

Cowdria polymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in an Escherichia coli host expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominant rproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived from rprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510 rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater.

Contribution: This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.


Keywords

Heartwater; vaccine epitopes; PBMC; lymphocyte proliferation assay; ELISA; ELISpot; cytokine qRT-PCR; flow cytometry

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