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Production of Mutant Based Epsilon Toxin (ETK) Vaccines

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Critique of Li et al. Article: A low-toxic site-directed mutant of Clostridium perfringens É›-toxin as a potential candidate vaccine against enterotoxemia


  • Donnelle Phillips

Vaccines improve the immune system of an animal or human by stimulating the production of antibodies to fight and combat bacteria, protozoan or viruses effectively creating immunity against disease (Salisbury 1997). Vaccines commonly contain weakened or dead strains of a virus, bacteria or protozoan which elicits the body’s immune system upon recognition to respond by producing antibodies to fight against the invading antigen. In effect, the vaccine introduces the disease or its causative agent into the body to gain a defence response; as a result if the disease enters the body in the future the body can produce the requisite antibodies to fight the disease faster, as it remembers measures taken to fight against the disease previously. Consequently, the time taken to recognize the disease, provide active measure against the disease and minimize any possible signs or symptoms associated with the causative virus, bacteria or protozoan. Building on this function of vaccines this essay critiques the study and subsequent paper of Li et. al. investigation into the production of mutant based epsilon toxin (ETK) vaccines which can provide protection against intoxication.

Costridium perfringes a Gram positive spore forming bacterium, is ubiquitously found in the environment and the intestines of humans and animals. Furthermore, it is commonly found in raw meat and poultry and is also linked to foodborne illnesses and food poisoning (Centers for Disease Control and Prevention 2014). Presently, there are five known strains of C. perfringes which produce a minimum of twelve known protein toxins (The Centre for Food Security and Public Health 2004). To this end, the epsilon toxin (ETX) is produced by the type B and D strains (The Centre for Food Security and Public Health 2004). Moreover, ETX is a pore forming protein which damages cell walls leading to potassium and fluid leakage from cells and it increases the permeability of the cell membrane resulting in ion imbalance; as such ETX has been suggested as a possible biological weapon (Stiles, et al. 2013). Arizona Department of Health (2004) speculates that in the event of a biological attack the epsilon toxin can be spread through food, water or by aerosolization. While all five strains have the capacity to infect wounds in any species; type B and type D have the ability to cause severe enteritis, in young foals, lambs and piglets, and enterotoxemia in young calves, lambs, goats, sheep and foals just to mention a few (Arizona Department of Health Services 2004). The toxin is also known to cause pulmonary edema and neurological symptoms including recumbence hyperesthesia, convulsions, paddling, dyspnea and loss of conciseness when intravenously injected into animals. They are few reported cases on the toxicity of ETX in humans as the type B and D strains rarely infect humans. However, evidence suggest that it can affect the G-402 and ACHN cell lines of the human kidney highlighting its possible toxicity to humans (Li, et al. 2013).

The epsilon toxin is secreted as an inactive prototoxin from the cytosol into the environment as a result of a 32 amino acid residue leader sequence. It is then activated by extracellular serine type proteases of the host such as trypsin or chymotrypsin; which subsequently results in the loss 10-13 amino terminal and 22 or 29 carboxy terminal residues depending on the protease used (Stiles, et al. 2013). Moreover, maximal activation results in the loss of 13N-terminal residues and 29 C-terminal residues producing the third most potent toxin of the clostridia class, loss of these two terminals results in the formation of a more acidic protein which possibly favours more productive receptor interactions (Hunter, et al. 1992). Bokori-Brown et al. (2011) states that the prototoxin can also become activated by C. perfringens λ – protease. When activated the toxin becomes relatively resistant to proteases in the gastrointestinal tracts of mammals (Stiles, et al. 2013).The effects of the toxin on the cell membranes lead to cell dysfunction, cell death and suffering in the host which can sometimes be fatal. Proteolysis induces a conformational change in the toxin facilitating homo-oligomerization of the activated toxin on the external surface of the cell (Stiles, et al. 2013). After binding to a cell ETX acts quickly.

Two groups of pore forming toxins exist, to this effect ETX is a beta pore forming toxin as it forms a beta-hairpin structure to facilitate membrane insertion (Stiles, et al. 2013). Moreover, the toxin has three domains with implications in the function of the toxin. The first domain, domain I-amino terminus, functions in receptor binding, the central domain or domain II functions in membrane insertion and channel formation (Stiles, et al. 2013). The final domain, domain III located at the carboxy terminus functions in proteolysis activation and act as monomer to monomer interaction sites. Subsequent to the loss of the C terminal peptide from the protoxin monomer-monomer interactions leads to homo-heptamer formation. ETX cause pore formation in cell membranes by detergent-resistant, cholesterol-rich membrane domains that promote aggregation of toxin monomers in homo-heptamers (Stiles, et al. 2013). Evidence from research highlights ETX forms transmembrane pores less than two nanometre in diameter that facilitates the passage of molecule 1kDa in size, increase intracellular chloride and sodium levels and decreased intracellular potassium levels. Furthermore, secondary effects of the toxin include cytoskeletal dysfunction which affects the integrity of cell monolayers (Stiles, et al. 2013).

The general layout and presentation of the paper was good and easy for readers to follow. However, the materials and methods should have occurred before the results and discussion to familiarise the readers with the methods used in the experiment. In doing this readers would have an idea of the type of results to be presented or expected. Furthermore, if presented earlier the procedure could be borne in mind in order to help readers understand the information presented in the results and discussion sections and the relevance of the information to the aim of the research. Additionally, if the methodology is placed earlier in the paper it helps readers to determine the extent of the internal validity of the study, as readers can determine for themselves the extent to which results can be interpreted accurately and with confidence with regards to the methods used to obtain the presented findings. Reliability, a necessary condition for validity, is related to consistency of results and the extent to which the research can be independently replicated by other researchers (Leacock, Warrican and Rose 2009). Based on the information provided the study is reliable and can be replicated independently by researchers with the relevant resources. In addition the use of graphs, tables and photographs of the gels used in the experiment are good means of presenting information. To this end, it presents data in a concise format allowing readers to easily identify pertinent information and reduces the possibility of readers being overwhelmed if this information was to be fully present using only text. It is also important to note that the use of the photos form the SDS-PAGE and the immunoblotting aids in reader visualisation and acts as a point of reference for readers when examining the results and discussion.

The introduction presents a good base for the readers but would have benefited from more in depth explanation on the mode of action and structure of ETX and more background information on mutant based vaccinations. Also a proper comparison of the proposed mutant based vaccine and how it would essentially function in contrast to the existing vaccine which only protects against enterotoxemia caused by the disease would have increased the reader’s knowledge about the topic and reinforce the importance of find a viable vaccine with low toxicity. It is important to note that the introduction provided supportive information from previous research about the amino acid residues present in the structure of ETX and their function. By extension the introduction also highlights the chosen sites for mutation in the toxin based and evidence also presented in the paper.

A brief synopsis of the methods used with reference to article presented by Li et al. are as follows; sited directed mutagenesis of ETX in the pTIG-trx plasmid, with the obtained sequence being confirmed using nucleotide sequence analysis. Following this the pTIG-mETXs were transformed into competent E. coli for expression which was also driven with a T7 promoter. The bacteria was then collected and purified through a combination of centrifugation, buffer washes, resuspension, sonification, chelation and elution. The purified protein were subsequently analysed using SDS-PAGE, with the highly purified proteins undergoing dialysis and concentration. The next step employed ELISA to identify the antigenicity of the mETXs and rETX using anti-rETX monoclonal antibodies. After electrophoresis the purified mETX protein were transfer from the SDS-PAGE gel using the western blot technique. The ELISA and western blot steps served to illicit the antigenicity of the mETX which both involving the use of goat anti-mouse IgG during incubation periods. A cell culture and cytotoxicity assay was conducted to determine rETX activity by defining its effects on MDCK cells. Three dimensional structure of two mETXs, mETXF199E and mETXH106P, were generated for structural analysis. The two mentioned not-toxic mETXs were used to vaccinate six week old female mice with the same dosage of antigen being given on days 17 and 38 a week later the mice were injected with active recombinant mETX and observed for 72hours. The following step involved the measurement of sera antibody titers. The final step of the experiment tested mice for passive protection against rETX.

The use of ELISA is a good means of detecting the presence of ETX and is one of the assays highlighted by the Arizona Department of Health Services for detection of the toxin due to its high detection rates (Stiles, et al. 2013). ELISA combines the specificity of antibodies with the sensitivity of simple enzyme assay by coupling the two, in the experiment it was us to detect the presence of antibodies which recognize ETX (Sino Biological Inc 2014). Additionally, the use of coloured tags indicate a positive reaction when the substrate interacts with the enzyme which also gives visual confirmation of the presence of the antigen or antibody which would have useful implications for this study. Furthermore, it is effective as it produces relatively fast results, which is especially relevant as samples can degrade over time, deterioration may also have implications for the levels of antibodies present. Liquid chromatography-mass spectrometry uses inmmunoaffinity beads to concentrate ETX or prototoxin from a complex matrix and is also a method commonly used to determine the presence of ETX and may be given consideration if the study is replicated (Stiles, et al. 2013). While ELISA and mass spectrometry are commonly used in the detection they do not determine if the toxin is biologically active. The use of titration was effective as it determined the amount and levels of antibodies required to neutralize the toxin. Random assignment to treatment groups is good as it gives each mouse the same change of being assigned to a group and reduces possibility of bias. However, as only three mice there were used in each group there are possible implications for the analysis of the results, as for experimental research the minimum recommended number is 15 participants for each group, as a sufficiently large sample size is required to produce results among variables that are significantly different (Leacock, Warrican and Rose 2009). A bigger sample size may also be effective in giving better credence to the obtained results for each treatment. The use of only female rats can have implications for the generalization of the results and by extension the validity of the study. Furthermore, all of the mice used in the experiment were six weeks in age this does not provide variation and as such does not account for the possible effects age can have on the function of the vaccine and results of the rETX challenge. Validity applies to the generalization of research and the vaccines is aimed for use in animals and humans and these populations contain both males and females, thus the use of one sex in the experiment have implications for generalization of results. The study may have also benefited from longer observation period after the mice were injected with the rETX. Despite the potential market for the vaccine including humans the research was conducted using mice due to ethical considerations. Additionally, the creation of pharmaceutical therapies require several animal experiments to be conducted using the treatment under before the ability for a clinical study using humans and the vaccine being offered to the public.

The result section has sub headings corresponding to the methods used which aids in easy identification for readers. Some of the significant findings of the research include the yield of six mETXs and up to 98% purity was obtained after purification measures. Additionally, the results showed that the toxin mutants retained the same antigenicity as the rETX. Four of the toxin mutants showed decreased cytotoxicity, while mETXS111Y and mETXS111YF199E showed a slight decrease in toxicity and higher toxicity when compared to rETX thus the researchers excluded the latter two from further analysis. Also of importance is immunization and the subsequent ELISA results of the anti-mETX antibody titres highlighted mETXH106P and mETXF199E showed similar titres and there was no significant difference between the two; but antibody titers also increased after booster immunization. The most significant results demonstrated that mice when challenged with active rETX with dosages up to 100 x LD50 survived. In contrast all with dosage of 500 x LD50 or 1000 x LD50 died which have implications for use of the vaccine and provides grounds for further research to obtain vaccines which have a better response to higher dosage of rETX. Investigation into passive protection showed anti-mETXH106P or anit-mETXF199E can completely neutralize a 10 x LD50 dose of activated rETX. The discussion highlighted supporting facts from various sources but there was limited discussion to certain findings of the paper. A possible suggestion is that the authors provide a more in depth discussion about their findings instead of a synopsis. The discussion may have also benefited from a comparison of the mutant based vaccine with the effectiveness of current ETX related treatments. Furthermore in depth explanations of the findings would help readers understand the significance of the findings and implication for future production and use of the tested vaccine. A significant finding highlighted in the discussion is that modifications of various amino acid residues result in varying cytotoxicity of the ETX. To this end, the researchers should have discussed why all of the obtained mETX were not tested in the immunization phase. A recommendation is the use of all of the obtained mETXs in the vaccination section of the experiment to determine the possibility of its use as a vaccine to provide immunity against the epsilon toxin.

The study did record some success in meeting its aims as two of the mETXs, mETXsH106P and mETXF199E as possible candidates for vaccines against the toxin as they showed strong immunogenicity and safety. However, mETXF199E still has toxicity, as a consequence the researchers have made a possible suggestion to improve the vaccination and lower toxicity. In concluding this article has led to the advancement of the body of knowledge pertaining to protection against ETX. The information from the study despite some limitations is a good read for persons interested in the topic. To this effect, it also provides a theoretical and empirical basis for further study about therapeutic measures to combat the epsilon toxin. It is important to note that it provides a preventative method against the toxin where as other treatments tend to target the ETX related diseases and related symptoms of the toxicity of the toxin, which some treatments delaying the onset of the toxin’s effects or delaying or preventing death (The Centre for Food Security and Public Health 2004).


Arizona Department of Health Services. 2004. Epsilon Toxin of Clostridium perfringens Bioterrorism Agent Profiles for Health Care Workers. August. Accessed March 20, 2015. http://www.azdhs.gov/phs/emergency-preparedness/documents/zebra-manual/zm-s5-epsilon-toxin.pdf.

Bokori-Brown, M, C G Savva, S P Ferandes da Costa, C E Naylor, A K Basak, and R W Titball. 2011. "Molecular basis of toxicity of Clostridium perfringens epsilon toxin." The FEBS Journal 278 (23): 4589-4601.

Centers for Disease Control and Prevention. 2014. CDC- Clostridium perfrines - Food Safety. January 29. Accessed March 20, 2015. http://www.cdc.gov/foodsafety/clostridium-perfingens.html.

Hunter, S E, I N Clarke, D C Kelly, and R W Titball. 1992. "Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli." Infection and Immunity 60 (1): 102-110.

Leacock, C J, S J Warrican, and G Rose. 2009. Research Methods for Inexperienced Researchers. Kingston: Ian Randle Publisher.

Li, Qing, Wenwen Xin, Shan Gao, Lin Kang, and Jinglin Wang. 2013. "A low-toxic site-directed mutant of Clostridium perfringens É›-toxin as a potential candidate vaccine against enterotoxemia." Human Vaccines & Immunotherapeutics 9 (11): 2386-2392.

Salisbury, David M. 1997. "Some Issues Related to the Practice of Immunization." International Journal of Infectious Diseases 1 (3): 119-124.

Sino Biological Inc. 2014. ELISA Principle. Accessed March 20, 2015. http://www.elisa-antibody.com/ELISA-Introduction/ELISA-Principle.

Stiles, G Bradley, Gillian Bartg, Holger Barth, and Michel R Popoff. 2013. "Clostridium perfringens Epsilon Toxin: A Malevolent Molecule for Animals and Man?" Toxins (Basel) 5 (11): 2138-2160.

The Centre for Food Security and Public Health. 2004. Epsilon Toxin of Clostridium perfringens. January. Accessed March 20, 23015. http://www.cfsph.iastate.edu/Factsheets/pdfs/epsilon_toxin_clostridium.pdf.

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