Finding a New Vaccine in the Ricin Protein Fold

Abstract

Previous attempts to produce a vaccine for ricin toxin have been hampered by safety concerns arising from residual toxicity and the undesirable aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain partner. We undertook a structure-based solution to this problem by reversing evolutionary selection on the 'ribosome inactivating protein' fold of RTA to arrive at a non-functional, compacted single-domain scaffold (sequence RTA1-198) for presentation of a specific protective epitope (RTA loop 95-110). An optimized protein based upon our modeling design (RTA1-33/44-198) showed greater resistance to thermal denaturation, less precipitation under physiological conditions and a reduction in toxic activity of at least three orders of magnitude compared with RTA. Most importantly, RTA1-198 or RTA1-33/44-198 protected 100% of vaccinated animals against supra-lethal challenge with aerosolized ricin. We conclude that comparative protein analysis and engineering yielded a superior vaccine by exploiting a component of the toxin that is inherently more stable than is the parent RTA molecule.

Folding domains within the ricin toxin A subunit as targets of protective antibodies

“…FGA12 is the only non-neutralizing MAb that we have identified to date that recognizes an epitope within folding domain 1. Interestingly, the FGA12 epitope lies within the exact exposed hydrophobic loop that was deleted by McHugh and colleagues in an effort to stabilize the recombinant derivative of RTA [21, 22]. While our data suggest an intriguing correlation between epitope structure and neutralizing activity, additional MAbs against each of these regions are needed to fully validate this conclusion.…”

“…The first is a recombinant derivative of RTA that contains a point mutation in the toxin’s active site, as well as a mutation in a region of RTA attributed to eliciting vascular leak syndrome [16]. The second is a recombinant form of RTA known as 1-33/44-198 that carries a deletion of an exposed 10 amino acid hydrophobic loop (T34-P43) within folding domain 1, as well as a truncation of all of folding domain 3 (A199 - F267) [20–22]. In both cases, however, RTA has been engineered in the absence of a fundamental understanding of the regions of the toxin that are critical in eliciting protective immunity.…”

“…RiVax is currently in Phase I clinical trials (E. Vitetta and J. Smallshaw, UT Southwestern, personal communication). The second RTA derivative being pursued as a vaccine was developed by the U.S. Army and is called RTA 1-33/44-198 [20–22]. This variant carries a deletion of an N-terminal proximal exposed loop region (T34-P43) as well as a truncation of the C-terminus (A199- F267).…”

Intradermal administration of RiVax protects mice from mucosal and systemic ricin intoxication

“…The use of recombinant RTA containing a 25 amino acid inactivating insertion plus alum was immunogenic and protective in mice, but residual catalytic activity makes it an unlikely candidate for a human vaccine [10]. Another vaccine developed by United States Army Medical Research Institute for infectious diseases (USAMRIID) utilizes a truncated RTA, which is structurally very stable, immunogenic and is a highly promising vaccine [11]. However, none of these vaccines eliminates the vascular leak-inducing site found in RTA and therefore, immunization could cause local tissue damage.…”

The Need for Continued Development of Ricin Countermeasures

“…USAMRIID has developed a recombinant RTA vaccine 1–33/44–198 (rRTA 1–33/44–198) (RV Ec ) produced in Escherichia coli [30–32]. Based on preclinical studies, including a pivotal repeated-dose toxicology study in New Zealand white rabbits conducted under GLP [33], this product was determined to have a reasonable safety profile for use in human studies.…”