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

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.

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

Figure 1

Characterization of the monomerized ricin vaccine. (A) SDS-PAGE analysis of the ricin purification and monomerization process (0.25 µg/lane) samples. Lane 1, Marker; Lane 2, Crude ricin; Lane 3, Purified ricin; Lane 4, Monomerized ricin. (B) UPLC chromatograms of alkylated-ricin and its purified subunits. 5 µL of the tested samples were injected into the UPLC columns (2.1 × 50 mm) and eluted at a flow rate of 0.4 mL/min. The chromatograms were monitored at 215 nm. 1. Alkylated-ricin preparation. 2. Alkylated-RTA. 3. Alkylated-RTB. (C) Catalytic activity assessment of ricin and monomerized ricin in a cell free system. The catalytic activities of purified ricin holotoxin (black line) and monomerized ricin (blue line) were determined using the transcription and translation (TnT) assay. Luminescence of untreated reticulocytes was considered as 100% protein synthesis. (D) In vitro activity of pure and alkylated ricin. Cultured HEK-293-AChE cells were incubated with increasing concentrations of pure (black line) and alkylated (blue line) ricin. The residual AChE activity in the culture medium was determined and expressed as the percent activity determined for untreated cells. (E) Toxicity following monomerized ricin administration to mice. Monomerized ricin (40 µg/kg body weight) was intraperitoneally administered to mice (n = 10), and body weights were determined at the indicated time points (blue line). Phosphate buffered saline (PBS) injected mice served as control (black line). Animals were observed for a 14 days period after alkylated ricin or PBS were injected.

Figure 2

Titer buildup in the horse serum after vaccination with monomerized ricin immunization. The reactivity profile of the antibodies elicited by immunization were determined by enzyme linked immunosorbent assay (ELISA, black curve) and by in vitro ricin neutralization assay (blue curve).

Figure 3

In vitro and in vivo efficacy of the anti-ricin F(ab’)₂-based antitoxin. (A) In vitro ricin neutralization. Ricin (2 ng/mL) was mixed with increasing concentrations of the antitoxin. The mixtures were added to cultured HEK-293-AChE cells, and the residual AChE activity in the culture medium was determined 18 h later. (B) Kaplan–Meier survival curves of mice intoxicated with ricin and subjected to anti-ricin antibody treatment. Mice intranasally intoxicated with ricin (7 µg/kg body weight) were not treated (black line; n = 14), treated intranasally at 24 h post exposure (blue line; n = 29), treated intravenously at 24 h post exposure (red line; n = 26), or treated intravenously at 48 h post exposure (green line; n = 38) with horse derived F(ab’)₂ anti-ricin antitoxin. Animals were observed for a 14-day period after ricin challenge.