Thermostable ricin vaccine protects rhesus macaques against aerosolized ricin: Epitope-specific neutralizing antibodies correlate with protection

Abstract

Ricin toxin (RT) is the second most lethal toxin known; it has been designated by the CDC as a select agent. RT is made by the castor bean plant; an estimated 50,000 tons of RT are produced annually as a by-product of castor oil. RT has two subunits, a ribotoxic A chain (RTA) and galactose-binding B chain (RTB). RT binds to all mammalian cells and once internalized, a single RTA catalytically inactivates all of the ribosomes in a cell. Administered as an aerosol, RT causes rapid lung damage and fibrosis followed by death. There are no Food and Drug Administration-approved vaccines and treatments are only effective in the first few hours after exposure. We have developed a recombinant RTA vaccine that has two mutations V76M/Y80A (RiVax). The protein is expressed in Escherichia coli and is nontoxic and immunogenic in mice, rabbits, and humans. When vaccinated mice are challenged with injected, aerosolized, or orally administered (gavaged) RT, they are completely protected. We have now developed a thermostable, aluminum-adjuvant-containing formulation of RiVax and tested it in rhesus macaques. After three injections, the animals developed antibodies that completely protected them from a lethal dose of aerosolized RT. These antibodies neutralized RT and competed to varying degrees with a panel of neutralizing and nonneutralizing mouse monoclonal antibodies known to recognize specific epitopes on native RTA. The resulting antibody competition profile could represent an immunologic signature of protection. Importantly, the same signature was observed using sera from RiVax-immunized humans.ricin | vaccine | monoclonal antibody | rhesus macaques | immunoprofiling R icin toxin (RT) is made by the plant Ricinus communis, which grows worldwide. RT can be easily prepared from pulverized castor beans and is very toxic even in crude form (1). Because of its prevalence and ease of preparation, RT is listed on the CDC Select Agent and Toxins list. RT consists a 32-kDa A chain (RTA) linked by a disulfide bond to a 34-kDa B chain (RTB) (2-4). RTA is a catalytic class II ribosome inactivating protein, RTB is a galactose-specific lectin. The LD 50 of RT varies according to the route of exposure. Administered as an aerosol, RT has an LD 50 of 5-15 μg/kg (5).Although both RTA and RTB are immunogenic, most experimental vaccines against RT have used some form of RTA; protection is mediated by antibodies. The leading vaccine candidates at this time are RVEc, developed by the Department of Defense (6, 7), and RiVax, developed at the University of Texas Southwestern (8-11). RiVax is a recombinant RTA with two mutations (V76M, Y80A) that eliminate both its enzymatic activity and its ability to induce vascular leak syndrome in humans (8, 12). The crystal structure of RiVax is virtually identical to that of native RTA, indicating that the two amino acid mutations have a minimal effect on the tertiary structure of the protein (13). The majority of the conformational epitopes should therefore be intact (13). The recombinant protein is a minimu...

Comparative Adjuvant Effects of Type II Heat-Labile Enterotoxins in Combination with Two Different Candidate Ricin Toxin Vaccine Antigens

“…We and others have recently reported that toxin-neutralizing titers generally lag behind total ricin-specific IgG levels by 1 to 2 weeks, a phenomenon that we note in Discussion (15,16). It was, therefore, not surprising that serum samples from day 17 were devoid of detectable in vitro TNA (data not shown).…”

“…This lag in TNA relative to total toxin-specific antibody levels has been noted in previous studies conducted in mice (16) and, very recently, in rhesus macaques (15). In the former study, groups of mice received RiVax, a modified anthrax protective antigen (PA), or a combination of the two.…”

Management of Castor Bean Seed-Associated Intraventricular Hemorrhage with Fresh Frozen Plasma: A Case Report

“…However, no certain antidote has been suggested to treat the patients. Since the ricin might be used as biological weapons, some efforts have been made to make a vaccine or antidote for ricin (15,16). It has been explained that ricin is a lectin (a carbohydrate-binding protein) and formed in the seeds of the castor oil plant.…”

“…It has been explained that ricin is a lectin (a carbohydrate-binding protein) and formed in the seeds of the castor oil plant. Researchers have evaluated ricin of the castor bean as vaccine candidate for safety of humans and animals against this compound in the United Kingdom and United States (15,16).…”

The potential of toxin-based drug delivery systems for enhanced nucleic acid therapeutic delivery

“…Mature (lethal) RT consists of a 267 residue RTAC and a 262 residue RTBC, held together by a disulphide bond between Cys 294 (on RTAC) and Cys 318 (on RTBC) [69]. RTAC attenuation is facile and has been undertaken to produce a potential anti-RT vaccine [70].…”