A heterologous prime/boost immunisation strategy protects against virulent E. ruminantium Welgevonden needle challenge but not against tick challenge
Introduction
Heartwater is a disease transmitted to wild and domestic ruminants by Amblyomma tick species. The causative agent of heartwater is Ehrlichia ruminantium, a rickettsial agent that is prevalent in sub-Saharan Africa and three islands in the Caribbean. Protective immunity to heartwater requires the activation of a cellular immune response. Four E. ruminantium open reading frames (ORFs) cloned in a DNA vaccine vector pCMViUBs, a vector known for cytotoxic T cell (CTL) induction, resulted in 100% protection in sheep when the animals were needle challenged with a virulent E. ruminantium stock [1]. Protection was therefore most likely conferred by CTL and CD4+ T helper one (TH1) lymphocytes. However, this vaccine was not protective when used in the field against a natural infected tick challenge [2]. We decided, therefore, to investigate new immunisation strategies to improve the immunogenisity and protective efficacy of our rDNA vaccine against heartwater.
rDNA priming followed by recombinant protein or recombinant modified viral boosting has been shown to improve immunity to several viral and bacterial diseases. Recombinant pox- and adenoviruses are the most frequently used viral vector antigen delivery systems. Protection against several pathogens, which are controlled primarily by a cellular immune response, has been demonstrated using the rDNA prime/modified viral boost strategy. These pathogens include: Mycobacterium tuberculosis (MTB) [3], Plasmodium falciparum, P. berghei, P. yoelii and P. knowlesi[4], [5], [6], [7], [8], [9], [10], Leishmania infantum[11], Schistosoma mansoni[12], classical swine fever virus [13] and human immunodeficiency virus [14], [15]. All these studies showed increased levels of protection that coincided with increased cytotoxic T cell responses and elevated levels of interferon gamma (IFN-γ) when compared to rDNA only immunisation experiments.
rDNA priming followed by recombinant protein boosting has also been used in attempts to improve immunity to bacterial diseases including heartwater. The first rDNA prime/recombinant protein boost immunisation strategy for the development of subunit vaccines against heartwater used the major antigenic protein (MAP1) as antigen [16]. The researchers were able to show that a rDNA prime/recombinant protein boost, using Quil A as adjuvant, improved survival in mice against a virulent E. ruminantium challenge, while immunisation with the recombinant protein alone did not induce protection at all. The observed protection also coincided with elevated levels of IFN-γ in the mice splenocytes. Similar results were also obtained in an attempt to improve vaccine strategies against MTB. A rDNA prime/recombinant protein boost using the ESAT6 gene as antigen not only improved the protection against lethal MTB but also improved lymphocyte proliferation and increased IFN-γ production [17].
In this study, we investigated whether a rDNA prime followed by either a r1H12 protein boost or a recombinant modified viral boost would induce improved protection against heartwater after a needle challenge or a natural field challenge.
Section snippets
E. ruminantium ORFs
Four E. ruminantium vaccine candidate genes were originally identified in a cosmid clone known as 1H12. These ORFs, designated Erum2540, Erum2550, Erum2580 and Erum2590 [1], were previously cloned into the DNA vaccine vector, pCMViUBs [2].
Preparation of recombinant lumpy skin disease virus (r1H12_LSDV)
Primer sets containing either BamHI or SalI restriction sites, were designed (Table 1) to facilitate directional cloning of the 1H12 ORFs into the pAFMCR shuttle vector obtained from the University of Cape Town Medical School, South Africa [18]. The ORFs were
Laboratory trial 1: rDNA prime (i.m. and i.d.) followed by either a r1H12_LSDV or r1H12 protein boost, compared with rDNA only immunisation
Sheep immunised with 3× 1H12 rDNA cocktail and sheep immunised with 2× 1H12 rDNA cocktail followed by 1× r1H12 protein boost all had a 100% survival rate. These animals did not have a critical rise in temperature (not higher than 40.6 °C) and only a few animals in each group developed early heartwater symptoms (Fig. 1). As expected, significant RI differences were obtained between the 1H12 inoculated groups (3× pCMViUBs_1H12 ORF cocktail (P < 0.001) and 2× pCMViUBs_1H12 ORF cocktail/1× r1H12
Discussion
Previously we reported that 100% of sheep immunised using a DNA immunisation strategy with pCMViUBs_1H12 ORF constructs were protected against a virulent needle challenge [1], while only 20% protection was observed when this same immunisation strategy was tested in the field [2]. To improve the protection obtained by the 1H12 rDNA vaccination strategy in the field, heterologous prime/boost immunisation was investigated. First, it was assessed whether priming with rDNA followed by boosting with
Acknowledgements
The authors thank Stephen Johnston (University of Texas Southwestern Medical Center, USA) for supplying the pCMViUBs vector and Anna-Lise Williamson (University of Cape Town Medical School, RSA) for supplying the LSDV virus and the pAFMCR shuttle vector. We thank Ms. H.C. Steyn for the screening of animals with the pCS20 test and the preliminary sequence data. This work was supported by the South African Department of Science and Technology LEAD biotechnology grant and the EU
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2019, VaccineCitation Excerpt :However, this was not the case in our study; some of the sheep that survived challenge did not show any significant IFN-γ production although one did. Previous heartwater challenge studies have also observed a lack of correlation between IFN-γ production and protective immunity [25,26,40] highlighting a need to identify additional correlates of protective immunity. In addition to IFN-γ responses, memory T cell responses were analysed and these responses were highly variable and also showed evidence of lack of correlation with protection as observed with the IFN-γ responses.