Abstract
Ricin, a highly lethal toxin derived from the seeds of Ricinus communis (castor beans) is considered a potential biological threat agent due to its high availability, ease of production, and to the lack of any approved medical countermeasure against ricin exposures. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this work was to generate anti-ricin antitoxin that confers high level post-exposure protection against ricin challenge. Due to safety issues regarding the usage of ricin holotoxin as an antigen, we generated an inactivated toxin that would reduce health risks for both the immunizer and the immunized animal. To this end, a monomerized ricin antigen was constructed by reducing highly purified ricin to its monomeric constituents. Preliminary immunizing experiments in rabbits indicated that this monomerized antigen is as effective as the native toxin in terms of neutralizing antibody elicitation and protection of mice against lethal ricin challenges. Characterization of the monomerized antigen demonstrated that the irreversibly detached A and B subunits retain catalytic and lectin activity, respectively, implying that the monomerization process did not significantly affect their overall structure. Toxicity studies revealed that the monomerized ricin displayed a 250-fold decreased activity in a cell culture-based functionality test, while clinical signs were undetectable in mice injected with this antigen. Immunization of a horse with the monomerized toxin was highly effective in elicitation of high titers of neutralizing antibodies. Due to the increased potential of IgG-derived adverse events, anti-ricin F(ab’)2 antitoxin was produced. The F(ab’)2-based antitoxin conferred high protection to intranasally ricin-intoxicated mice; ~60% and ~34% survival, when administered 24 and 48 h post exposure to a lethal dose, respectively. In line with the enhanced protection, anti-inflammatory and anti-edematous effects were measured in the antitoxin treated mice, in comparison to mice that were intoxicated but not treated. Accordingly, this anti-ricin preparation is an excellent candidate for post exposure treatment of ricin intoxications.
Highlights
Ricin, a type II ribosome inactivating protein (RIP) derived from the seeds of Ricinus communis, consists of two polypeptide subunits (A and B) linked by a disulfide bond
ELISA- and neutralizing-antibody titers were reached following immunization with both native or monomerized ricin (2.56 × 105 vs. 4.8 × 105 ELISA units; 7.68 × 104 vs. 9.6 × 104 neutralizing units, respectively). In line with these findings, hyperimmune sera collected from rabbits immunized with either monomerized or native ricin, conferred high level protection in vivo; when mice were treated intramuscularly (i.m.) with the rabbit antisera 24 h prior to i.m. ricin challenge, the doses which conferred 50% protection (PD50 ) were 0.8 and 1.1 μL/mouse, respectively. These experiments suggest that immunization of a horse with monomerized ricin vaccine would be as effective as immunization with native toxin in elicitation of neutralizing anti-ricin antibodies, while the neutralized monomer-based antigen would possess much greater safety margins
bronchoalveolar lavage fluids (BALFs) of antitoxin-treated and non-treated ricin-intoxicated mice, In addition to overall protein level, we have previously demonstrated that increased pulmonary edema respectively)
Summary
A type II ribosome inactivating protein (RIP) derived from the seeds of Ricinus communis (castor beans), consists of two polypeptide subunits (A and B) linked by a disulfide bond. Anti-ricin antibodies may be elicited following vaccination of various animal species, including mice [8], rabbits [9], monkeys [10], horses [11] and sheep [12]. A toxoid-based vaccine (formaldehyde-inactivated ricin) was shown to induce high titers of protective antibodies both in rabbits [4] and in sheep [12]. Anti-ricin preparations were reported to elicit potent toxin neutralization in vitro and in vivo following horse immunization with an RTA/RTB chain construct, in which the native inter-chain linking domain has been replaced by a non-cleavable linker [11]
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