Ricin Intoxication: History
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Contributor:
Christine Rasetti-Escargueil
,
Arnaud Avril

Ricin toxin is a disulfide-linked glycoprotein (AB toxin) comprising one enzymatic A chain (RTA) and one cell-binding B chain (RTB) contained in the castor bean, a Ricinus species. Ricin inhibits peptide chain elongation via disruption of the binding between elongation factors and ribosomes, resulting in apoptosis, inflammation, oxidative stress, and DNA damage, in addition to the classically known rRNA damage. Ricin has been used in traditional medicine throughout the world since prehistoric times. Because ricin toxin is highly toxic and can be readily extracted from beans, it could be used as a bioweapon (CDC B-list). 

  • antitoxin
  • antibodies
  • ricin
  • small-molecule inhibitors

1. Diversity and Structure of Ricin

1.1. Ricin Diversity and Structure

Contrary to ricin E, ricin D is present in all castor bean seeds. For instance, ricin E is not found in R. communis cv. zanzibarensis, which is known to contain only the ricin D isoform, but it is found in R. communis cv. Carmencita. Moreover, differences in post-translational modifications (e.g., N-glycosylation), which can also impact toxicity, have been described depending on the cultivar and origin of the seed from which ricin is isolated. The ricin E has been defined as a hybrid form of ricin because it is composed of a ricin-like A chain, the N-terminal half of the B chain and an RCA-like C-terminal half of the B chain [1][2]. Initially, it was considered that ricin E was present in small-grain castor beans, but a more recent publication questioned this [1]. “Small seeds” refer to cultivars where 100 seeds weigh less than 20 g.
The bi-chain nature of the ricin structure was demonstrated in the early 1970s, when ricin was found to be composed of an active chain (A chain) and a binding chain (B chain) linked by a disulfide bond [3][4]. The primary sequences of both ricin chains and their holotoxin structures at 2.8 Å resolution were solved soon after [5][6].
The refined ricin structure at 2.5 Å revealed that the ricin A chain is a globular protein consisting of 267 amino acids organized into eight α-helices and eight β-strand structures. The ricin B chain consists of 262 amino acids having two homologue domains, each containing a lactose binding site [7][8][9][10]. It was then discovered that the ricin A chain can be cross-linked to the ribosomal proteins L9 and L10e [11][12] and that its active site involves key residues such as Tyr80, Tyr123, Glu177, Arg180, and Trp211. Ribosomal adenine is deemed to be trapped between Tyr80 and Tyr123 in a π stacking interaction and then protonated by Arg180, promoting the formation of an oxocarbenium moiety on the ribose [13][14].

1.2. Ricin Enzymatic Activity

By disrupting the binding between elongation factors and ribosomes, ricin inhibits peptide chain elongation, avoiding the elongation-factor-dependent GTPase activity [15][16]. Similar plant proteins have been identified to inhibit protein synthesis, holding a comparable polypeptide chain to the A chain of ricin: they are called “ribosome-inactivating proteins” (RIPs) [17][18]. Finally, in 1987, Endo et al. discovered the enzymatic nature of the ricin A chain, which cleaved the N-glycosidic bond of an adenine residue in rats, making RNA highly susceptible to hydrolysis [19]. Furthermore, ricin was found to release adenine from rRNA, DNA, and poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase, an enzyme involved in DNA repair [20]. It was also highlighted that, in tandem with protein synthesis inhibition and apoptosis, ricin could act directly on DNA in several cellular models.

1.3. Ricin Cellular Uptake, Routing, and Toxicity

Early studies demonstrated that the ricin interaction with the cell starts with the binding of the B chain to galactosyl residues of both glycolipids and glycoproteins on the cell surface, allowing access to the endosomal compartment [21]. Ricin is subsequently internalized, using various endocytic pathways, to reach the Golgi apparatus and intoxicate the cell. Ricin first penetrates the cell via clathrin-dependent endocytosis [22]. Ricin is then delivered to early endosomes. In parallel, ricin is recycled back to the cell surface or delivered to lysosomes via late endosomes for proteolytical degradation [23][24]. Eiklid et al. showed that cell death induced by ricin is directly proportional to the ricin concentration. The velocity of ricin activity is much greater than the speed of re-synthesis/repair of ribosomes, and ricin’s half-life is sufficient to inactivate all ribosomes in the cells. These observations suggest that one molecule of active ricin that reaches its substrate could be enough to kill one cell [25]. In addition to the disruption of protein synthesis, it was discovered that ricin, and related toxins, may be retrogradely transported across neuronal processes, paving the way for innovative research in neurobiology [26]. Ricin is one of the most toxic plant toxins discovered with an IC50 of less than 0.1 to 1 pM. Considering their polynucleotide-depurinating activity, RIPs may have a wider toxic action such as the oxidative stress potential facilitation. This suggests that ricin is able to drive the induction of more than one cell death pathway [27].
However, the cellular mechanism of action that kills the cell is quite complex. Several studies show that the inhibition of protein synthesis is not always correlated with long-term ricin toxicity. It is now admitted that, in addition to the rRNA damage, ricin can induce apoptosis, inflammation, oxidative stress, and DNA damage. At present, the correlation between these processes is under investigation. In addition to expanding investigations on intoxication processes via intracellular transport, knowledge of ricin’s action on cells, including its ability to enter the cytosol, can be applied to treat infectious diseases or for even wider therapeutic purposes [28].

2. Development of Anti-Ricin Vaccines

Generally, the best approaches to prevent a pathology are prophylaxis and, in particular, vaccination. Because of the cost and the balance between the benefit and the risk of a vaccine, vaccination is generally not recommended for rare diseases or toxins. In the context of toxins, post-exposure vaccination is generally not efficient. In studies with rhesus macaques, at 4 h post-intoxication, administration of a neutralizing mAb protected 5/5 monkeys, but only 1/5 at 12 h post-intoxication [29]. Considering the risk of bioterrorism and due to the potential short window for treatment, a vaccine may be suitable in some contexts, and anti-ricin vaccines are currently being studied. In a normal context, anti-ricin vaccination could be limited to people at high risk of exposure (scientists, first responders, or soldiers) to prevent laboratory accidents.
Previous studies have shown the A chain to be more immunogenic than the B chain [30]. Thus, two recombinant vaccines based on immunization with the RTA subunit are currently in phase 1 and 1B development, namely, RVEcTM (US Army Medical Research Institute of Infectious Diseases: USAMRIID, Frederick, MD, USA) and RiVax® (the University of Texas Southwestern Medical Center, Dallas, TX, USA) [31].
Rivax is an alum-adjuvanted subunit vaccine candidate that may prevent death and injury from any route of exposure to RT. A major advantage of this vaccine is that RiVax® is a thermostabilized vaccine candidate that can be stored at room temperature for extended periods and is stable up to 40 °C. Such stability is essential for stockpiling and in a military context because soldiers may be engaged everywhere in the world and because the cold chain may not be respected. RiVax® is composed of a modified form of the A chain of RT that removes the biological activity of the protein while still retaining its shape to trigger an effective antibody response. One mutation disrupts the ribotoxic site (Y80A), and the other one disrupts the vascular-leak-syndrome-inducing site (V76M) [32][33]. RiVax® is administered as an intramuscular injection on two or three occasions, resulting in the adaptive immune system mounting an antibody response. In a clinical study, all volunteers with anti-RTA antibodies also had ricin-neutralizing antibodies in their sera 2 weeks after the third vaccination. It was observed that the level of neutralizing antibodies was not related to the dose of the vaccine given, but almost all volunteers receiving the lowest vaccine dose did not develop an antibody response. An individual whose antibody had the most robust neutralizing activity received their third vaccination 112 days after the second vaccination, whereas all the other volunteers received theirs 28–42 days after the second vaccination. A passive protection assay was realized in a mouse model with the sera of the volunteers that developed neutralizing antibodies. The highest doses tested (62.5 μg and 25 μg) protected the mice from 5 LD50 of ricin (or 2 μg), whereas lower doses (12.5 μg and 5.0 μg) did not. The RiVax® antigen has been shown to be safe in two phase 1 studies in humans. None of the 15 volunteers experienced grade 3 or 4 symptomatic toxicity in this study. Two experienced grade 2 toxicities. The toxicities were those often associated with i.m. injections of approved vaccines. The development of RiVax® is pursuing its approval under the Animal Rule. Future steps will include pivotal animal efficacy studies (to demonstrate potency in animals) and phase 2 clinical studies in humans (to confirm its safety and correlate immune markers of protection with outcomes from animal studies).
A recombinant ricin vaccine from E. coli (RVEc™) was developed at the USAMRIID (Frederick, MD, USA) and assessed in an FDA-sponsored phase 1a clinical trial. RVEcTM is a truncated derivative of RTA that lacks the hydrophobic carboxy-terminal region (residues 199–267) as well as a small hydrophobic loop in the N-terminus (residues 34–43), resulting in a molecule with increased solubility and thermal stability [34][35][36]. RVEcTM does not contain mutations that directly inactivate the active site of RTA, but the removal of both segments results in an inactive molecule devoid of enzymatic activity and a reduction in or elimination of its ability to cause a vascular leak, as demonstrated in experimental models. RVEcTM is adjuvanted with Alhydrogel®. Volunteers received 3 injections at 4-week intervals with 20 or 50 μg of vaccine (n = 10 for each group), and a group of volunteers received a single dose of 100 μg. RVEc™ was safe and well-tolerated at all doses. The most common adverse events were pain at the injection site and headache. Of the 10 subjects who received a single 100 μg dose, 2 developed elevated creatine phosphokinase levels, which resolved without sequelae. Anti-ricin IgG titers of 1:500 to 1:121,500 were observed using ELISA assays in subjects immunized with 20 µg or 50 μg. A total of 50% of them produced neutralizing anti-ricin antibodies measurable via TNA. Four subjects in the 50 μg group received a single booster dose of RVEc™ 20–21 months after the initial dose. The single booster was safe and well-tolerated, resulting in no serious adverse events and significantly enhanced immunogenicity of the vaccine in human subjects. Each booster recipient developed a robust anamnestic response with ELISA anti-ricin IgG titers of 1:13,500 to 1:121,500 and neutralizing antibody titers of 1:400 to 1:3200.

This entry is adapted from the peer-reviewed paper 10.3390/toxins15020100

References

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