**Generation of Highly Efficient Equine-Derived Antibodies for Post-Exposure Treatment of Ricin Intoxications by Vaccination with Monomerized Ricin**

**Reut Falach 1, Anita Sapoznikov 1, Ron Alcalay 1, Moshe Aftalion 1, Sharon Ehrlich 1, Arik Makovitzki 2, Avi Agami 2, Avishai Mimran 2, Amir Rosner 3, Tamar Sabo 1, Chanoch Kronman 1 and Yoav Gal 1,\***


**\*** Correspondence: yoavg@iibr.gov.il; Tel.: +972-8-9381479

Received: 25 October 2018; Accepted: 8 November 2018; Published: 12 November 2018

**Abstract:** Ricin, a highly lethal toxin derived from the seeds of *Ricinus communis* (castor beans) is considered a potential biological threat agen<sup>t</sup> 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.

**Keywords:** ricin; vaccine; antitoxin; RTA; RTB; reduction; alkylation

**Key Contribution:** In this study, we demonstrate that treatment of mice at clinically relevant time points with anti-ricin F(ab')2-based antitoxin derived from a horse immunized with neutralized ricin, conferred exceptionally high protection against a lethal intranasal ricin challenge.
