CYTOTOXICITY AND EFFICACY OF THE PLANT-DERIVED EtxD ANTIGEN

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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 μg/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. It has been shown in the study that a transiently expressed EtxD protein can be efficiently purified from tobacco to a high purity and yield by using just two main steps after the initial extraction. Up to 49,85% product yield (based on the initial recombinant EtxD protein concentration) could be recovered after the final step, as visualised on the SDS-PAGE. The recombinant EtxD protein was recovered to purity, as judged by the fact that a Coomassie stained SDS-PAGE has a single band. The results suggest that the transiently expressed EtxD protein may be efficiently purified from N. benthamiana extracts. The purification steps incorporated in the study also suggest that this purification scheme has the potential to be scaled-up. It has also been determined that it is possible to produce a relevant and less toxic Pulpy Kidney Disease vaccine in plants by means of Nicotiana sp. transient expression via the recombinant A. tumefaciens. Based on the results, the plant-derived purified EtxD protein needs to be trypsin-activated and formulated with a strong adjuvant, such as the DCA immune stimulant, to obtain full protection in mice. Further work needs to be undertaken to establish the dose response and technoeconomic model for production of this candidate vaccine. The current study thus demonstrates the feasibility of producing a safe and potent subunit vaccine for Pulpy Kidney Disease vaccine. This is the first reported case of such an achievement with this particular disease.

THESIS COMPOSITION

In five chapters, this thesis describes a doctoral study aimed at efficiently producing a Pulpy Kidney Disease vaccine in N. benthamiana plants. Chapter 1 discusses the clinical importance of the Epsilon toxin responsible for the Pulpy Kidney Disease in economically important livestock. The chapter also provides information on the molecular biology of the Ɛ-toxin; the effects of the Epsilon toxin on MDCK cells; the effects of the toxin on animals; tissue, evidence of neurotoxicity; the crystal structure of the toxin; pore formation by Epsilon toxin; Pulpy Kidney Disease vaccine production and its challenges; and the plant-derived biopharmaceutical product. The chapter concludes with the introduction of tobacco as an expression host and Agrobacterium mediated transformation. Chapter 2 presents the methods and data of experiments relating to the expression feasibility and biochemical characterisation of a recombinant Epsilon toxin (EtxD) produced transiently as a soluble protein in N. benthamiana leaves. Chapter 3 informs on the methods used and the data obtained from experiments relating to the purification of the recombinant plant-derived EtxD protein in N. benthamiana plants, as well as the biochemical characterisation that confirms the presence and levels of protein. Chapter 4 presents the methods used and data obtained from experiments relating to the in vivo and in vitro studies undertaken to investigate the suitability of the plant-derived purified EtxD protein as an antigen for Pulpy Kidney Disease vaccine production, the evaluation of the toxicity of the plant-derived EtxD protein and the testing of the efficacy of the plant-derived Pulpy Kidney Disease vaccine. Chapter 5 summarises and provides perspective on the study followed by all the references that are cited within this thesis.

ACKNOWLEDGEMENTS

To Jehovah, thank You very much for the divine interventions and all the blessing you have provided through this journey. A heartfelt gratitude to my supervisors, Dr Rachel Chikwamba, Dr Ereck Chakauya, Dr Tsepo Tsekoa and Dr Michael Crampton, for their leadership and support that has made this achievement possible. DCA Lab for the in vivo studies undertaken to investigate the suitability of the plant-derived purified EtxD protein as an antigen for Pulpy Kidney Disease vaccine production I would like to also express my gratitude to my husband and family for their unfailing support and encouragement. Gogo nasiMelane, this is for you.

Clinical importance of the Epsilon toxin from C. perfringens

According to Robinson et al. (2017), the Clostridium genus encompasses more than 80 species that form a diverse group of rod-shaped, Gram positive bacteria that have the ability to form spores. These organisms are principally obligate anaerobes, although some species are able to survive in the presence of trace amounts of oxygen (Bokori-Brown et al., 2011; Stiles et al., 2013). Clostridia are omnipresent bacteria that is found in soil and water, as well as in decomposing animal and plant matter. In addition, some Clostridia species can be found in the gastrointestinal tract of humans and animals where they form part of the normal gut flora. However, under certain circumstances some of these species are able to cause severe diseases in humans and domestic animals as a result of a variety of toxins being produced (Stiles et al., 2013). Clostridium perfringens is one of the most pathogenic species in the Clostridium genus as it is able to produce at least 17 toxins (Alouf, 2006; Bokori-Brown et al., 2011). Depending on their ability to produce the four typing toxins (α, β, ε and ι-toxins), C. perfringens strains are classified into five toxinotypes (Table 1.1) (Petit et al., 1999; Bokori-Brown et al., 2011; Stiles et al., 2013; Jemal., 2016). In addition to the typing toxins, the bacterium is able to produce a number of toxins not used for typing, such as β2, δ, ĸ, μ, γ, ν, ө and enterotoxin (Stiles et al.,2013). As bacterial toxins often act in concert, causing virulent effects, their individual significance and roles in disease can be difficult to assess. The Epsilon toxin is produced by the C. perfringens toxinotypes B and D, which also produces the β-toxin, is the aetiological agent of dysentery in new-born lambs. It is also associated with enteritis and enterotoxaemia in goats, calves and foals (Berger, 2016). C. perfringens type D affects mainly sheep (including lambs) that are on rich diets, but it can also cause infection in goats and calves (Bokori-Brown et al., 2011). The most important factor that initiates the disease is the disruption of the microbial balance in the gut as a result of overeating. This leads to the passage of large amounts of undigested carbohydrates from the rumen into the intestine. Here, C. perfringens is then able to proliferate in large numbers and produce the Ɛ-toxin. The overproduction of this toxin causes increased intestinal permeability, facilitating the entry of the toxin into the bloodstream and then spreading to various organs, including the brain, lungs and Kidneys, which causes severe oedema (Bokori-Brown et al., 2011). While infection of the central nervous system results in neurological disorders, a further effect is sudden death (Finnie, 2003; Bokori-Brown et al., 2011). The US Government Centre for Disease Control and Prevention considers the toxin to be a potential bio-warfare or bio-terrorism agent (Berger, 2016). The use of biological weapons in conventional warfare has been banned by the Biological and Toxic Weapons Convention, initiated by the USA in 1972. Western states are particularly concerned about the availability of toxins to terrorist groups. The fact that a 50% dose (LD50) of Epsilon toxin in mice (50 ng/kg) is lethal (Berger, 2016), indicates the potential for using this toxin as a bio-terrorist weapon and highlights the need to understand the molecular basis of the toxicity so that an effective vaccine can be developed.

Impact of the sheep industry in South Africa

Sheep production in South Africa is a significant contributor to food security and clothing. In addition, the industry adds considerable value to the country’s economy. For these reasons and to ensure the industry’s healthy growth, efficient and affordable vaccines are required to prevent diseases such as Pulpy Kidney Disease (Bath et al., 2016). As Pulpy Kidney Disease is the most important disease that constrain small-ruminant livestock farming growth (Jemal et al., 2016). Furthermore, this supports the contention that animal disease control requires significant continued support and lack of access to veterinary support including vaccines is a major challenge to farming SMEs. Hence, the development of affordable, easily accessible, safe and quality vaccine such as plant-based Pulpy Kidney Disease vaccine is required. The current OBP price for a Pulpy Kidney Disease vaccine is R155, 48c/100 ml excluding tax. The constant monitoring of sheep after grazing for the disease and revaccination makes the vaccination process expensive to small-ruminant livestock farmers and hinders their growth (Bath et al., 2016). Sheep farming is practiced throughout South Africa, although it is concentrated in the more arid parts of the country such as the Northern, Eastern and Western Cape, the Free State and Mpumalanga. The industry encompasses approximately 8 000 commercial sheep farms and about 5 800 communal farmers. The number of sheep in South Africa is estimated to number 28,8 million. Sheep farmers are represented by organisations such as the Dorper Sheep Breeders’ Society of South Africa and Merino SA (Snyman, 2014). The Dorper is a highly successful South African mutton breed developed specially for the more arid areas of the country. Its excellent carcass qualities in terms of conformation and fat distribution generally qualify it for top classification. Other mutton breeds that also produce wool are Damara, Meatmaster, Ille de France, Dormer, Suffolk, Van Rooy and Vandor (Snyman, 2014). The gross value of mutton production is dependent on the price and quantity of meat produced. Over the past ten years the average gross production value amounted to R3,4 billion per year. This value has increased continuously over the years (Snyman, 2014).

South Africa’s competitiveness of the sheep industry

South Africa is a net importer of mutton as it only produces approximately 83% of its domestic consumption. Given the current maize surplus and a low world maize price, with grain prices expected to remain under pressure, there should be a further improvement in profitability by supplementary feeding. Domestic mutton prices are expected to move with the normal seasonal price trend, which declines during October and increase during the braai months. It is therefore expected that prices will strengthen due to strong consumer demand during this period (Pieter, 2017).

Molecular biology of the Epsilon toxin

The Ɛ-toxin gene, Etx, is located on plasmids in both toxinotypes B and D (Bokori-Brown et al., 2011). In toxinotype B isolates, the Etx gene is carried on a 65 kb plasmid that may also carry the cpb2 gene for the β2-toxin (Sayeed and McClane, 2010; Bokori-Brown et al., 2011), while the cpb gene that encodes the β-toxin resides on a separate plasmid. In the toxinotype D isolates, the Etx gene is present on plasmids ranging from 48 to 110 kb (Sayeed and McClane, 2010; Bokori-Brown et al., 2011). Interestingly, the larger plasmids have been found to carry up to three different toxin-encoding genes (Etx, cpe and cpb2) (Sayeed and McClane, 2010; Bokori-Brown et al., 2011). A common theme in both toxinotypes is the association of the Etx gene with insertion sequences. The transposable element IS1151 has been found upstream of the Etx gene in plasmids from both toxinotypes, although in opposite orientations (Miyamoto et al., 2008; Bokori-Brown et al., 2011). This association has led to speculation about possible virulence gene mobilisation and exchange between plasmids. Support for this hypothesis was provided by the identification of circular transposition intermediates containing IS406-Etx-IS1151 (Bokori-Brown et al., 2011). These findings have implications for the evolution of C. perfringens and help to explain how some plasmids carry multiple toxin genes. Additional evidence for genetic exchange among toxinotypes is provided by the finding that the tcp locus, required for conjugation (Bokori-Brown et al., 2011), is present in some Etx plasmids from both toxinotype B and D isolates (Sayeed and McClane, 2010). Hughes et al. (2007) demonstrated the conjugative transfer of an Etx plasmid from a toxinotype D to a type A isolate, thus essentially converting type A to type D, both genotypically and phenotypically (Hughes et al., 2007; Bokori-Brown et al., 2011). In all strains, Ɛ-toxin is expressed with a signal sequence of 32 amino acids that direct an export of the prototoxin from C. perfringens (Bokori-Brown et al., 2011). Sequencing of EtxB and EtxD revealed only two nucleotide differences in the open reading frames. The first change, at position 762, does not result in an amino acid substitution. The second change, at position 962, results in a substitution from serine, in EtxB, to tyrosine in EtxD (Bokori-Brown et al., 2011). The upstream regions of the EtxB and EtxD genes are not identical and have different putative -10 and -35 promoter regions (Bokori-Brown et al., 2011). This suggests that the expression of these genes may be regulated in different ways in type B and D strains of C. perfringens. This possibility is supported by the observation that the strain from which the EtxD gene was isolated (NCTC 8346) produced ten times more Ɛ-toxin than the strain from which the EtxB gene was isolated (NCTC 8533) (Havard et al., 1992). The relatively inactive secreted prototoxin of 296 amino acids (32,9 kDa) is converted to the fully active mature toxin by a proteolytic cleavage in the gut lumen. This is either by digestive proteases of the host, such as trypsin and chymotrypsin (Bhown and Habeerb, 1993), or by C. perfringens k-protease (Minami et al., 1997; Bokori-Brown et al., 2011). Proteolytic activation of the toxin can also be achieved in the laboratory by controlled enzyme digestion (Bokori-Brown et al., 2011). Depending on the protease, proteolytic cleavage results in the removal of 10 to 13 amino-terminal amino acids and 22 to 29 carboxy-terminal amino acids (Bhown and Habeerb, 1993; Bokori-Brown et al., 2011). Maximal activation of the toxin occurs with a combination of trypsin and chymotrypsin, resulting in the loss of 13 N-terminal residues and 29 C-terminal residues (see Figure 1.1).

Effect of the Epsilon toxin on MDCK cells

The effects of Ɛ-toxin on several cell lines have been tested on cultured cells over the past few decades to identify a suitable in vitro model for the study of the Epsilon toxin. The Madin- Darby Canine Kidney (MDCK) cell line of epithelial origin, derived from the distal collecting tubule, was initially identified to be toxin sensitive by a microscopic examination of intoxicated cells (Bokori-Brown et al., 2011). Cytotoxicity assays on a further 11 Kidney cell lines of animal origin failed to identify additional cell lines sensitive to the toxin (Bokori-Brown et al., 2011). Cytotoxicity assays on 17 human cell lines (originating from the Kidney, brain, skin, bone, respiratory and intestinal tracts) identified the Caucasian renal leiomyoblastoma (G-402) cell line to be toxin-sensitive, although to a lesser extent than the MDCK cell line (Shortt et al., 2000). In MDCK cells, the dose of Ɛ-toxin needed to kill 50% of cells is reported to be 15 ng/ml, while the MDCK cells that have Epsilon toxin in their system undergo morphological changes that include swelling and formation of membrane blebs (Bokori-Brown et al., 2011; Takeyana et al., 2015). The rapid death of cells exposed to the toxin results in the formation of a large membrane complex on the target cell surface (Donelli et al.,2003; Bokori-Brown et al., 2011) leading to pore formation, an efflux of K+ and an influx of Na+ and Clions (Petit et al., 2001). In addition, cytotoxicity is temperature and pH-dependent (Lindsay, 1996) and is potentiated by EDTA (Bokori-Brown et al., 2011). Recently, the cytotoxic effect of Ɛ-toxin was demonstrated in a highly differentiated murine renal cortical collecting duct principal cell line, mpkCCDcl4 (Chassin et al., 2007). These cells retain the specific ion transport properties of the distal collecting duct cells from which they are derived (Chassin et al., 2007; Bokori-Brown et al., 2011). According to Buxton (1990), in mpkCCDcl4 cells, a toxin-induced intracellular Ca2+ rise and an ATP depletion-mediated cell death occurred even under conditions that prevented toxin oligomerisation and thus pore formation. Some primary cells are also susceptible to the toxin. For example, guinea pig peritoneal macrophages exposed to the toxin show a blistering of the nuclear membrane, an ill-defined chromatin and a swollen cytoplasm without structure. Mixed glial primary cell cultures, isolated from mice brains, are also toxin-sensitive (Soler- Jover et al., 2007). Primary cultures of mice cerebellar cortex identified granule cells are also targeted and affected by the Epsilon toxin, leading to membrane severing, Ca2+ influx and glutamate efflux (Lonchamp et al., 2010). Primary cultures of the human renal tubular epithelial cells also showed a toxin-induced swelling of cells and the formation of membrane blebs (Fernandez-Miyakawa et al., 2010).

Table of Contents :

• ABSTRACT
• ACKNOWLEDGEMENTS
• ABBREVIATIONS AND SYMBOLS
• Chapter INTRODUCTION
  • 1.2 Impact of the sheep industry in South Africa
  • 1.2.1 South Africa’s competitiveness of the sheep industry
  • 1.3 Molecular biology of the Epsilon toxin
  • 1.4 Effect of the Epsilon toxin on MDCK cells
  • 1.5 Effects of the Epsilon toxin on animals and tissues
  • 1.6 Evidence for neurotoxicity
  • 1.7 Crystal structure of the Ɛ-toxin
  • 1.8 Pore formation by Epsilon toxin
  • 1.9 Pulpy Kidney Disease vaccine production and challenges
  • 1.10 Plant-derived biopharmaceutical products
  • 1.11 Tobacco as an expression host
  • 1.12 Agrobacterium-mediated delivery
  • 1.13 Downstream processing
  • 1.14 The development of expression vectors
• 2 INTRODUCTION
  • 2.1 Pulpy Kidney Disease
  • 2.2 Materials and method
  • 2.3 Results
  • 2.4 Discussion
  • 2.5 Conclusion
• Chapter
  • 3.1 Introduction
  • 3.2 Materials and methods
  • 3.3 Results
  • 3.4 Discussion
  • CYTOTOXICITY AND EFFICACY OF THE PLANT-DERIVED EtxD ANTIGEN
  • ABSTRACT
  • 4. INTRODUCTION
  • 4.1 Current production and formulation methods of the Pulpy Kidney Disease vaccine
  • 4.2 Materials and methods
  • 4.4 Results
  • 4.5 Discussion
  • 4.6 CONCLUSION
  • SUMMARY AND PERSPECTIVE
  • 5.1 SUMMARY
  • 5.2 Limitation and drawbacks of the product
  • 5.3 FUTURE RESEARCH
  • References
  • Appendix A

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