No abstract

Ricin, isolated from the castor bean plant Ricinus communis, is included on the Centers for Disease Control and Prevention Category B list of bioterrorism agents, indicating that the toxin is moderately easy to disseminate and could result in moderate morbidity rates. This study evaluated two promising recombinant ricin subunit vaccines, one made using an Escherichia coli codon-optimized gene and the other using a yeast codon-optimized gene in E. coli-based fermentations. Rabbits were vaccinated four times over a period of 6 months and challenged with ∼10 to 30 times the median lethal dose of aerosolized ricin. All unvaccinated control rabbits were either found dead or humanely euthanized within 30 h postchallenge, while the rabbits vaccinated with either vaccine survived the exposure without adverse clinical signs. When the protective antibody responses were analyzed, no significant difference was seen between the two vaccines. However, there was a significant difference in the immune response over time for both vaccines tested. Although clinical pathology was unremarkable, significant histological lesions in the control animals included fibrinonecrotic pneumonia, acute necrotizing lesions in the upper respiratory tract, and necrotizing lymphadenitis in the lymph nodes draining the upper and lower respiratory tract. Vaccine-treated rabbits exhibited resolving lesions associated with ricin exposure, namely chronic inflammation in the upper respiratory tract and lungs, fibrosis, type II pneumocyte hyperplasia, and bronchiolitis obliterans. This study confirmed the safety and efficacy of two recombinant ricin subunit vaccines in rabbits, offering potential protection to warfighters and select populations.

Ricin is a potent toxin and potential bioterrorism weapon for which no specific licensed countermeasures are available. We report the safety and immunogenicity of the ricin vaccine RVEc™ in a Phase 1 (N=30) multiple-dose, open-label, non-placebo-controlled, dose-escalating (20, 50, and 100μg), single-center study. Each subject in the 20- and 50-μg dose groups (n=10 for each group) received three injections at 4-week intervals and was observed carefully for untoward effects of the vaccine; blood was drawn at predetermined intervals after each dose for up to 1 year. RVEc™ was safe and well tolerated at the 20- and 50-μg 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, two developed elevated creatine phosphokinase levels, which resolved without sequelae. No additional doses were administered to subjects in the 100-μg group. Immunogenicity of the vaccine was evaluated by measuring antibody response using the well standardized enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). Of the subjects in the 20- and 50-μg dose groups, 100% achieved ELISA anti-ricin IgG titers of 1:500 to 1:121,500 and 50% produced neutralizing anti-ricin antibodies measurable by 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. Future studies will attempt to optimize dose, scheduling, and route of administration. This study is registered at clinicaltrials.gov (NCT01317667 and NCT01846104).

No abstract

Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A-chain (RTA), which is an N-glycosidase enzyme, and the B-chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1-33/44-198 protein, herein denoted RVEa™) expressed in Escherichia coli using a codon-optimized gene was shown to be non-toxic, stable, and protective against a ricin challenge in mice. Here, we describe the process development and scale-up at the 12 L fermentation scale, and the current Good Manufacturing Practice (cGMP)-compliant production of RVEc™ at the 40 L scale. The average yield of the final purified bulk RVEc™ is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEc™ was >99% pure by three HPLC methods and SDS-PAGE. The intact mass and peptide mapping analysis of RVEc™ confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc™ was immunoprotective against lethal ricin challenge and elicited neutralizing anti-ricin antibodies in 95-100% of the vaccinated mice.