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.
