Expression and Purification of an efficacious recombinant Pulpy Kidney subunit vaccine made in N. Benthamiana

Abstract

Epsilon toxin (Etx) produced by Clostridium perfringens type D is responsible for a fatal Enterotoxemia (Pulpy Kidney Disease) in economically significant livestock such as sheep and goats. The only practical means of controlling this disease is by immunisation and avoiding the circumstances that are conducive to its occurrence. All currently available Pulpy Kidney Disease vaccines are based on a formalinised toxoid of Clostridium perfringens (Welchii) type D. This is either as an alum-precipitate, oil-emulsion formulation of the whole cell culture or a bacterial culture filtrate (Deepika, 2010). The current vaccine has several drawbacks. First, it is generally accepted that its chemical inactivation with formalin is difficult to standardise. Second, the classical methods of detoxification usually alter the overall protein structure in a random manner. Consequently, the immunogenicity of this type of a vaccine is decreased substantially. Third, there is a narrow range in balancing the detoxification (the strength of formalin commonly used is 1%) and the immunogenicity of the vaccine (Robertson et al. 2011). Alternatives to formalin inactivation have been proposed because of these challenges. These include ectopically expressing the mutated toxoids in other gram-positive microorganisms. Apparently, plants provide a genuine alternative for the expression of an immunogenic and non-toxic vaccine for the Pulpy Kidney Disease. Thus, in this study, the hypothesis was that the EtxD protein could be transiently expressed efficiently in Nicotiana benthamiana plants via deconstructed viral vectors, targeting the EtxD protein expression into the apoplast or the cytosol. This resulted in the investigation of the following six research topics: 1. The feasibility of producing the Epsilon toxin recombinantly in N. benthamiana. 2. The role of subcellular targeting in the observed level of expression and accumulation of the target protein. 3. The methods of purification that could be explored to recover and purify the EtxD protein that was transiently expressed recombinantly in the N. benthamiana leaves. 4. The suitability of the plant-derived purified EtxD protein as an antigen for Pulpy Kidney Disease vaccine production. 5. An evaluation of the toxicity of the plant-derived EtxD protein. 6. Testing the efficacy of the recombinant plant-derived EtxD antigen. In this context, the EtxD gene, accession number AY858558, and the genetic region from the NCBI and EMBL databases was targeted for this study since the EtxD gene is well-reported in published data and is similar to the EtxD-gene strain produced in South Africa. The targeted gene was then codon-optimised to be expressed in N. benthamiana plants and chemically synthesised by GeneArt. The codon-optimised EtxD gene was directly cloned into an Icon-deconstructed vector and vacuum infiltrated via an Agrobacterium- mediated transfer. Leaves of N. benthamiana were transfected by vacuum infiltration to deliver the EtxD gene transiently into the apoplast and the cytosol respectively. The plant-derived EtxD protein was then isolated from the plant matrix and biochemically analysed by SDS-PAGE, N-terminal peptide sequencing and Western Blot analysis. The EtxD protein was visible as a 34 kDa protein band on an SDS-PAGE gel. The protein band was isolated, and the sequence was confirmed by N-terminal sequencing, as well as by Western Blot analysis using a secondary polyclonal guinea pig anti-EtxD antibody. The plant-derived EtxD protein was then quantified by ELISA and thereafter the expression levels were established at 380 mg/kg fresh weight when targeted to the apoplast, and 300 mg/kg fresh weight when targeted to the cytosol. The apoplast plant-made EtxD protein was purified using a two-step chromatography method, namely ion exchange and size exclusion, with a 50% recovery of the EtxD protein on the final step of purification. To investigate the suitability of the plant-derived purified EtxD protein as an antigen for the Pulpy Kidney Disease vaccine, the toxicity of the EtxD protein was evaluated and the efficacy of the derived EtxD protein was tested. For these, both in vitro and in vivo studies were conducted. The LD50 studies on mice revealed that the plant-derived EtxD protein was slightly toxic, which correlated with the IC50 results on MDCK cells. For the animal-challenge results, two formulations of vaccines were prepared from the recombinant antigen EtxD protein and administered intravenously to mice. The formulations that contained the plant-derived EtxD protein that were not activated by trypsin were unable to protect mice against the Epsilon toxin challenge. This indicates that the Epsilon toxin in the purified plant extract was not immunogenic. When the plant-derived EtxD protein was treated with trypsin, inactivated with formalin and formulated with the adjuvant, alum, it was also non-protective. However, the formulation containing the plant-derived EtxD protein and Disease Control Africa (DCA) immune stimulant was protective. These findings indicated that the plant-derived Epsilon toxin is a viable recombinant antigenic vaccine when formulated with the immune stimulant DCA. In conclusion, this study has demonstrated that tobacco is a suitable host for the production of the EtxD protein. The ELISA results of the infiltrated tobacco leaf samples have demonstrated the successful expression of the 34 kDa EtxD protein together with glucan-endo-1, 3-beta-glucanase of about 25 kDa. The cytosol targeted strategy generated the lowest EtxD protein production at 300, 200 and 10 ?g/kg fw of the protein. For large scale-production of the EtxD protein, transient expression targeting to the apoplast is preferable because of the high yield of protein per fresh leaf weight achieved in this study.