A. Pretorius

A heterologous prime/boost immunisation strategy protects against virulent E. ruminantium Welgevonden needle challenge but not against tick challenge.

Heterologous prime/boost immunisation strategies using the Ehrlichia ruminantium 1H12 pCMViUBs_ORFs [Pretorius A, Collins NE, Steyn HC, Van Strijp F, Van Kleef M, Allsopp BA. Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames. Vaccine 2007;25(12):2316-24] were investigated in this study. All the animals immunised twice with a recombinant (r) DNA cocktail of four 1H12 pCMViUBs_ORFs followed by a r1H12 protein and those immunised 3x with 1H12 plasmid rDNA showed 100% protection against a virulent E. ruminantium Welgevonden needle challenge. In addition, 90% of the sheep immunised twice with rDNA and boosted with r1H12 lumpy skin disease virus (LSDV) survived. Only the lymphocytes isolated from the r1H12 protein boost group showed specific proliferation and increased interferon (IFN)-gamma expression. In contrast, only 20% protection was obtained in animals immunised with the rDNA prime/r1H12 protein boost when subjected to natural tick challenge in the field. Thus this heterologous prime/boost immunisation strategy had not conferred any significant protection against a field challenge.

A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20

Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCR TaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838-843]. The pCS20 quantitative real-time PCR TaqMan probe was compared to the currently used pCS20 PCR and PCR/32P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCR TaqMan probe was the most sensitive assay detecting seven copies of DNA/mul of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/32P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCR TaqMan probe assay was the most sensitive and can be performed within 2h it is an effective assay for epidemiological surveillance and monitoring of infected animals.

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.

Development of Improved Vaccines for Heartwater

Abstract: Heartwater is a tick‐borne disease of ruminants which causes major economic losses for domestic livestock owners throughout sub‐Saharan Africa and the Caribbean. It is caused by the intracellular rickettsia Ehrlichia (formerly Cowdria) ruminantium and the only commercially available vaccination procedure is over 50 years old. It involves infecting animals with cryopreserved sheep blood containing virulent E. ruminantium organisms, followed by antibiotic treatment when fever develops. Experimental attenuated, inactivated, and nucleic acid vaccine procedures have been investigated over the last half‐century, but none of them has yet been particularly successful. We have developed two new experimental vaccines, a live attenuated vaccine and a nucleic acid vaccine. The attenuated vaccine was developed by continuous passage of E. ruminantium organisms of the virulent Welgevonden isolate in a continuous canine macrophage‐monocyte cell line. After more than 50 passages the cultures produced no disease when inoculated into mice or sheep, and the inoculated animals were 100% immune to a subsequent lethal homologous needle challenge. The nucleic acid vaccine is based on four E. ruminantium genes from a genetic locus involved in nutrient transport. A cocktail of all four genes, cloned in a DNA vaccine vector and used to immunize sheep, engendered 100% protection against a subsequent lethal needle challenge with the homologous isolate and with each of five different virulent heterologous isolates. Sheep immunized with this cocktail were also exposed to a field challenge in a heartwater‐endemic area and few animals survived. This suggests that the local E. ruminantium genotypes were different from any which were administered by needle challenge, or that needle challenge is not a good model for tick challenge in the field.

Virus-specific CD8+ T-cells detected in PBMC from horses vaccinated against African horse sickness virus

African horsesickness (AHS) is an infectious but noncontagious viral disease affecting all species of Equidae. The recall immune response of AHSV naïve horses immunised with an attenuated African horsesickness virus serotype 4 (AHSV4) was characterised using immune assays including ELISPOT, real-time PCR (qPCR) and flow cytometry. The recall immune response detected in PBMC isolated from three inoculated horses showed an upregulation of circulating B lymphocytes that correlated with elevated IL-4 mRNA expression indicative of humoral immunity, but reduced frequency of CD4⁺ cells. In addition to the expected antibody response, an increase in CD8⁺ cells was also detected. Although these CD8⁺ cells may be CTL, the role of these cells in immunity against AHSV still has to be determined.

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 interferon-gamma (IFN-gamma) 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-gamma production was evaluated in vitro using lymphocyte proliferation and ELISPOT assays. All five recombinant proteins induced proliferation of immune PBMCs and IFN-gamma 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-gamma 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-gamma production is likely to be involved in conferring protective immunity against heartwater.

Sequencing of a 15-kb Ehrlichia ruminantium clone and evaluation of the cpg1 open reading frame for protection against heartwater.

Abstract: A 1.2 kb polymorphic fragment from the Gardel isolate of Ehrlichia (formerly Cowdria) ruminantium was used to isolate a 15kb clone from the E. ruminantium Welgevonden LambdaGEM‐11 library. This clone, WL2EL1, was subcloned and sequenced. Eight open reading frames (ORFs) were identified. The ORF in WL2EL1 which contained the Welgevonden homologue of the 1.2 kb polymorphic fragment was designated Cowdria polymorphic gene 1 (cpg1). The cpg1 ORF was cloned into pCMViUB, a genetic vaccine vector. Mice and sheep were immunized with pCMViUB/cpg1 by intramuscular injection and gene gun inoculation. Although all of the immunized mice died, there was a trend for mice that received larger amounts of pCMViUB/cpg1 DNA to survive longer. Four out of five sheep immunized with the construct survived lethal challenge.

Studies of a polymorphic Ehrlichia ruminantium gene for use as a component of a recombinant vaccine against heartwater

A previously identified polymorphic Ehrlichia ruminantium gene, Erum2510, was investigated to determine its ability to induce protective immunity in ruminants following two different DNA immunisation strategies; DNA-only and a DNA prime/recombinant protein (rprotein) boost immunisation. The DNA-only vaccine was also compared to a cocktail of three polymorphic E. ruminantium (Welgevonden) open reading frames (ORFs) adjacent to Erum2510 in the genome. Weak protection was observed in animals immunised with the pCMViUBs_Erum2510 construct alone, while none of the animals immunised with the DNA cocktail were protected. In contrast, all five animals immunised using a DNA prime/rprotein boost strategy survived challenge, thereby indicating that Erum2510 is a good candidate for inclusion in a recombinant vaccine against heartwater. One drawback of using polymorphic genes is a possible lack of cross-protection between genotypes, therefore the genetic diversity of Erum2510 was investigated to establish the degree of polymorphism among different E. ruminantium stocks. Three distinct genotypes were identified indicating that if this gene is used as a vaccine (prime/boost strategy) the vaccine should include a representative Erum2510 gene from each genotype.

Immune gene expression profiling of PBMC isolated from horses vaccinated with attenuated African horsesickness virus serotype 4.

Development of African horsesickness (AHS) subunit vaccines will have to include a rational approach that uses knowledge of how the virus interacts with the host immune system. The global in vivo immune response induced by attenuated AHSV serotype 4 in horses was characterised using transcriptome sequencing. PBMC were collected with 24h intervals for four days after inoculation and four days after a second boost, 21 days later. Transcriptome data were normalised to the day 0 naïve transcriptome and up- or down-regulated immune genes identified using the CLC workbench. Peak expression was observed 24h after each inoculation. Innate immunity was up-regulated after both inoculations and was characterised by type-1 interferon activation via the RIG-1/MDA5 pathway and the up-regulation of complement cascade components. After the second boost an adaptive immune response could be identified that included the production of cytokines indicative of T helper (Th)1, Th2 and Th17 responses.

Transcriptomic analysis of Ehrlichia ruminantium during the developmental stages in bovine and tick cell culture

The use of bioinformatics tools to search for possible vaccine candidates has been successful in recent years. In an attempt to search for additional vaccine candidates or improve the current heartwater vaccine design, a genome-wide transcriptional profile of E. ruminantium (Welgevonden strain) replicating in bovine endothelial cells (BA886) and Ixodes scapularis embryonic tick cells (IDE8) was performed. The RNA was collected from the infective extracellular form, the elementary bodies (EBs) and vegetative intracellular form, reticulate bodies (RBs) and was used for transcriptome sequencing. Several genes previously implicated with adhesion, attachment and pathogenicity were exclusively up-regulated in the EBs from bovine and tick cells. Similarly, genes involved in adaptation or survival of E. ruminantium in the host cells were up-regulated in the RBs from bovine cells. Thus, it was concluded that those genes expressed in the EBs might be important for infection of mammalian and tick host cells and these may be targets for both cell and humoral mediated immune responses. Alternatively, those exclusively expressed in the RBs may be important for survival in the host cells. Exported or secreted proteins exclusively expressed at this stage are ideal targets for the stimulation of cytotoxic T-lymphocyte (CTL) immune responses in the host.