Comparative Aspects of Ricin Toxicity by Inhalation.

The pathogenesis of ricin toxicity following inhalation has been investigated in many animal models, including the non-human primate (predominantly the rhesus macaque), pig, rabbit and rodent. The toxicity and associated pathology described in animal models are broadly similar, but variation appears to exist. This paper reviews the published literature and some of our own unpublished data and describes some of the possible reasons for this variation. Methodological variation is evident, including method of exposure, breathing parameters during exposure, aerosol characteristics, sampling protocols, ricin cultivar, purity and challenge dose and study duration. The model species and strain used represent other significant sources of variation, including differences in macro- and microscopic anatomy, cell biology and function, and immunology. Chronic pathology of ricin toxicity by inhalation, associated with sublethal challenge or lethal challenge and treatment with medical countermeasures, has received less attention in the literature. Fibrosis may follow acute lung injury in survivors. There are advantages and disadvantages to the different models of pulmonary fibrosis. To understand their potential clinical significance, these factors need to be considered when choosing a model for chronic ricin toxicity by inhalation, including species and strain susceptibility to fibrosis, time it takes for fibrosis to develop, the nature of the fibrosis (e.g., self-limiting, progressive, persistent or resolving) and ensuring that the analysis truly represents fibrosis. Understanding the variables and comparative aspects of acute and chronic ricin toxicity by inhalation is important to enable meaningful comparison of results from different studies, and for the investigation of medical countermeasures.

Establishment of a Novel Oral Murine Model of Ricin Intoxication and Efficacy Assessment of Ovine Ricin Antitoxins.

Ricin, produced from the castor beans of Ricinus communis, is a cytotoxin that exerts its action by inactivating ribosomes and causing cell death. Accidental (e.g., ingestion of castor beans) and/or intentional (e.g., suicide) exposure to ricin through the oral route is an area of concern from a public health perspective and no current licensed medical interventions exist to protect from the action of the toxin. Therefore, we examined the oral toxicity of ricin in Balb/C mice and developed a robust food deprivation model of ricin oral intoxication that has enabled the assessment of potential antitoxin treatments. A lethal oral dose was identified and mice were found to succumb to the toxin within 48 h of exposure. We then examined whether a despeciated ovine F(ab')2 antibody fragment, that had previously been demonstrated to protect mice from exposure to aerosolised ricin, could also protect against oral intoxication. Mice were challenged orally with an LD99 of ricin, and 89 and 44% of mice exposed to this otherwise lethal exposure survived after receiving either the parent anti-ricin IgG or F(ab')2, respectively. Combined with our previous work, these results further highlight the benefit of ovine-derived polyclonal antibody antitoxin in providing post-exposure protection against ricin intoxication.

Production, Characterisation and Testing of an Ovine Antitoxin against Ricin; Efficacy, Potency and Mechanisms of Action.

Ricin is a type II ribosome-inactivating toxin that catalytically inactivates ribosomes ultimately leading to cell death. The toxicity of ricin along with the prevalence of castor beans (its natural source) has led to its increased notoriety and incidences of nefarious use. Despite these concerns, there are no licensed therapies available for treating ricin intoxication. Here, we describe the development of a F(ab')2 polyclonal ovine antitoxin against ricin and demonstrate the efficacy of a single, post-exposure, administration in an in vivo murine model of intoxication against aerosolised ricin. We found that a single dose of antitoxin afforded a wide window of opportunity for effective treatment with 100% protection observed in mice challenged with aerosolised ricin when given 24 h after exposure to the toxin and 75% protection when given at 30 h. Treated mice had reduced weight loss and clinical signs of intoxication compared to the untreated control group. Finally, using imaging flow cytometry, it was found that both cellular uptake and intracellular trafficking of ricin toxin to the Golgi apparatus was reduced in the presence of the antitoxin suggesting both actions can contribute to the therapeutic mechanism of a polyclonal antitoxin. Collectively, the research highlights the significant potential of the ovine F(ab')2 antitoxin as a treatment for ricin intoxication.

Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters.

DNA vaccines which expressed the Hc fragment of the Clostridium botulinum type F neurotoxin (BoNT/F Hc) fused to a signal peptide downstream of four different eukaryotic promoters were prepared. Subsequently, the immunogenicity of the DNA vaccines and protection afforded in mice against challenge with 10(4) MLD of type F botulinum toxin was evaluated. The DNA vaccine containing the human ubiquitin gene (UbC) promoter induced the highest BoNT/F Hc-specific antibody concentration following two intramuscular immunisations and afforded 90% protection against challenge. The results from this study indicate that the selection of promoter used in DNA vaccination studies may be of importance in designing optimised vaccines.

DNA vaccination protects against botulinum neurotoxin type F.

A DNA vaccine was constructed which expressed the binding domain of Clostridium botulinum neurotoxin serotype F fused to a signal peptide. Three intra-muscular doses fully protected Balb/c mice against 10(4) MLD of serotype F toxin. Priming of the immune response by DNA vaccination followed by a single booster with type F binding domain protein resulted in high levels of antibody against the binding domain. This study demonstrates the utility of DNA vaccination for protection against botulinum neurotoxin type F and indicates that a prime-boost regimen could be an efficient method of generating antibody for passive immune therapy in cases of botulism involving serotype F toxin.

Vaccination against type F botulinum toxin using attenuated Salmonella enterica var Typhimurium strains expressing the BoNT/F H(C) fragment.

The utility of the htrA, pagC and nirB promoters to direct the expression of the carboxy-terminal (H(C)) fragment of botulinum toxin F (FH(C)) in Salmonella enterica var Typhimurium has been evaluated. Only low levels of serum antibody were induced after immunisation, and some protection against botulinum toxin type F was demonstrated after oral immunisation of mice with two doses of any of these recombinant Salmonella. Immunisation with two doses of recombinant Salmonella expressing FH(C) from the htrA promoter gave the greatest protection, against up to 10,000 mouse lethal doses of botulinum toxin type F. These results demonstrate the feasibility of an orally delivered vaccine against botulinum toxin type F.

Anti-immunoglobulin responses to IgG, F(ab')2, and Fab botulinum antitoxins in mice.

Side effects to botulinum antitoxins, including anaphylaxis and serum sickness, are common. This is due to the immunogenicity of the antitoxin, which can be measured by the production of anti-immunoglobulin antibodies. An ideal botulinum antitoxin would elicit a minimal production of anti-immunoglobulin antibodies from a patient, aiding its safety. To investigate the immunogenicity of different immunoglobulin fragments, whole IgG, F(ab')2 and Fab botulinum antitoxins were administered to mice by either the intravenous or intramuscular route. The production of anti-immunoglobulin antibodies was measured over time after a single dose of antitoxin, and the anti-immunoglobulin antibodies isotyped. When administered by the intramuscular route, Fab showed significantly lower immunogenicity than IgG, while F(ab')2 had an immunogenicity that was intermediate between the two. When administered by the intravenous route there was no significant difference in immunogenicity between IgG and F(ab')2 antitoxins, although Fab antitoxin had a significantly lower immunogenicity than either IgG or F(ab')2. IgG antitoxin was significantly more immunogenic by the intramuscular route than by the intravenous route. Sheep IgG had a lower immunogenicity than goat IgG in mice. There was no significant difference in immunogenicity between the two dosing routes for either F(ab')2 or Fab antitoxin. The anti-antibodies were predominantly IgG1, suggesting a strong Th2 bias to the anti-antibody response. In all cases, Fab represents the least immunogenic form of antitoxin.