**1. Introduction**

Ricin, 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. The B subunit (RTB) is a lectin, which binds to galactose residues on the cell surface, enabling toxin internalization into the cells. The catalytically active A subunit (RTA) is translocated into the cytoplasm, where it depurinates a conserved adenine residue within the 28S ribosomal RNA of the 60S subunit, leading to irreversible inhibition of protein synthesis and ultimately to cell death [1]. Classified as a Category B agen<sup>t</sup> by the U.S. Centers for Disease Control and Prevention (CDC), ricin is considered a potential bioterror agen<sup>t</sup> mainly due to its high availability and ease of preparation [2]. Ricin toxicity depends on the route of exposure, inhalational and parenteral being highly fatal. Although prophylactic anti-ricin vaccines are being developed [3], the only post-exposure measure found effective against ricin intoxications in pre-clinical settings, is passive immunization with anti-ricin neutralizing antibodies [4–7]. 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 variety of ricin immunogens were employed to elicit neutralizing antibody responses against ricin. 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vivo following horse immunization with an RTA/RTB chain construct, in which the native inter-chain linking domain has been replaced by a non-cleavable linker [11]. Vaccination of animals using the native ricin toxin emulsified in adjuvant was also effective in the elicitation of high titers of neutralizing antibodies [13].

As part of ongoing efforts to develop novel ye<sup>t</sup> safe vaccination strategies that will induce high titers of ricin neutralizing antibodies, we established a method for ricin subunit-based immunization following irreversible monomerization of the toxin to its RTA and RTB constituents. The antigen was prepared by treating the toxin with a reducing agen<sup>t</sup> to sever the inter-subunit covalent bond, and then alkylating the monomeric toxin subunits to prevent their re-dimerization, thereby generating a stable monomerized ricin preparation for animal immunization with substantially reduced toxicity.

In the present study, the efficacy potential of the monomerized ricin vaccine was demonstrated in rabbits, after which the antigen, which was produced at large amount, was thoroughly characterized. The antigen, which was used for vaccinating the horse, was not only safe, but also elicited high titers of highly potent neutralizing antibodies against ricin. Passive immunization with the F(ab')2-based antitoxin conferred high protection against a lethal intranasal ricin challenge at clinically relevant treatment time points following intoxication, and displayed significant anti-inflammatory and anti-edematous effects.
