**2. Results**

#### *2.1. Elicitation of Anti-Ricin Antibodies Following Rabbit Immunization with Monomerized Ricin*

To determine whether the deactivation of ricin by monomerization, affects its ability to elicit neutralizing antibodies, we compared the anti-ricin antibody titers elicited by immunization either with the monomeric antigen or with native holotoxin. To this end, rabbits were immunized at 4-week intervals, with increasing antigen doses reaching up to 100 μg antigen/rabbit, and hyperimmune sera collected at 16 weeks after the first immunization dose were characterized. As seen (Table 1) high ELISA- and neutralizing-antibody titers were reached following immunization with both native or monomerized ricin (2.56 × 10<sup>5</sup> vs. 4.8 × 10<sup>5</sup> ELISA units; 7.68 × 10<sup>4</sup> vs. 9.6 × 10<sup>4</sup> neutralizing units, respectively). In line with these findings, hyperimmune sera collected from rabbits immunized with either monomerized or native ricin, conferred similarly 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 sugges<sup>t</sup> 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.

**Table 1.** Anti-ricin antibody titers and protective doses of anti-ricin antisera elicited in immunized rabbits.


a Values present the average ± standard error (SEM) of 5–6 rabbits. b Mice were treated with the respective antisera 24 h prior to challenge with 2LD50 (30 μg/kg) ricin. Values present the average ± standard error (SEM) of 3 mice or 6mice(treatedwithantiseraharvestedfromrabbitsimmunizedwithnativeormonomerizedtoxin,respectively).

#### *2.2. Characterization of the Monomerized Ricin Vaccine*
